Methods of inhibiting T-cell mediated B-cell antibody production using anti-CD27 antibodies

ABSTRACT

Human antibodies immunospecific for human CD27 are capable of blocking CD27 binding to its ligand CD70 and neutralizing bioactivity of CD27 including, but not limited to, CD27 intracellular signaling, T-cell proliferation and activation, B-cell proliferation and differentiation, plasmablast formation and alleviation of antibody responses, stimulation of tumor cells by CD70, and the production of soluble mediators from T and B-cells. The antibodies are useful in diagnosing or treating CD27 activity associated diseases and conditions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/790,144, filed 2 Jul. 2015, now U.S. Pat. No. 9,683,046, issued 20Jun. 2017, which is a divisional of U.S. application Ser. No.13/835,518, filed 15 Mar. 2013, now U.S. Pat. No. 9,102,737, issued 11Aug. 2015, which claims the benefit of U.S. Provisional Application Ser.No. 61/611,332, filed 15 Mar. 2012, the entire contents of which areincorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to human antibodies to the CD27 proteinand their uses and, more particularly, human antibodies to human CD27protein and their use in treating inflammatory disorders.

Related Art

CD27 is a type I transmembrane protein and member of the TNF receptorsuperfamily (TNFSF27) expressed as a surface antigen on a majority of Tcells, natural killer cells and antibody secreting plasma and memoryB-cells. CD70 is a cytokine, also called tumor necrosis factor ligandsuperfamily member 7 (TNFSF7), and the cognate ligand for CD27. TNFSFligand-receptor interactions are able to regulate T-dependent B-celldifferentiation (Jacquot S. 2000 Immunol Res. 21(1):23-30) and induceapoptotic cell death in different cells.

CD27:CD70 ligation results in activation of canonical and non-canonicalNF-kβ signaling pathways that in turn stimulates B- and T-cellproliferation, plasma cell differentiation and subsequent antibodysecretion (Yamamoto, H. 1998 J Immunol. 161(9): 4753-9). CD27co-stimulation with OX40, 4-1BB also promotes the survival of activatedT cells (Croft, M. 2003 Cytokine Growth Factor Rev. 14(3-4): 265-73),thereby regulating a number of effector and memory T cells and controlsT cell function directly by promoting production of cytokines, such asIL-4 and IFNgamma, or modulating T-cell responses to the actions ofother cytokines, such as IL2 and IL-12.

Studies in both humans and animals suggest an important role of theCD27:CD70 pathway in various immune-related diseases, including systemiclupus erythematosus (SLE) (Doerner T Lupus 2004 13(5):283-9), rheumatoidarthritis (Tak, P P et al. 1996 clin Immunol Immunopathol 80(2): 129-38)and multiple sclerosis (Hintzen R Q et al. 1991 J Neuroimmunol35(1-3):211-7). On the other hand, CD70 has been reported to beexpressed to varying degrees on malignant B cells and the CD70:CD27complex is able to mediate an antitumor response by activating antitumorimmunity and reducing tumor growth (Borst J, Hendriks J and Xiao Y.2005. Curr Opin Immunol. 17(3):275-81). CD27 may also control theaccumulation of CD4+ and CD8+ T-cells at sites of infection (Hendrickset al. 2000 Nature Immunol 1, 433-440).

CD70 is not expressed on normal non-hematopoietic cells. CD70 expressionappears to be temporally restricted to antigen-activated T- and B cellsand its expression is down-regulated when antigenic stimulation ceases.Evidence from animal models suggests that CD70 may contribute toimmunological disorders such as, e.g., rheumatoid arthritis (Brugnoni etal., 1997 Immunol. Lett. 55:99-104), psoriatic arthritis (Brugnoni etal., 1997, Immunol. Lett. 55:99-104), and lupus (Oelke et al., 2004,Arthritis Rheum. 50:1850-60). In addition to its potential role ininflammatory responses, CD70 is also expressed on a variety oftransformed cells including lymphoma B cells, Hodgkin's andReed-Sternberg cells, malignant cells of neural origin, and a number ofcarcinomas.

Agonist CD27 binding antibodies described in WO2008/051424 (Univ. SouthHampton) are noted as useful for promoting T-cell immunity and suchantibodies have a binding epitope which causes them to be unaffected(not inhibited) by CD70.

While studies in rodents involving alteration of CD27 and/or CD70 havedemonstrated potentially important roles of this receptor ligandinteraction, there is a need to provide human antibodies specific forhuman CD27 and other CD27:CD70 interaction blocking agents that canexert a clinically useful cytotoxic, cytostatic, or immunomodulatoryeffect on CD27-expressing cells, particularly without exertingundesirable agonist effects on CD27-expressing cells in the absence ofCD70. Such compounds may be useful therapeutic agents in modulating thedevelopment of neoplastic cells or immune disorders that are mediated byCD27-expressing cells.

SUMMARY OF THE INVENTION

The present invention provides human CD27 binding, monoclonal antibodiescapable of blocking activities associated with CD27-CD70 interaction oncells, tissues, or organs in a host subject. Amino acid sequences ofexemplary CD27 binding monoclonal antibodies are provided which areencoded by nucleic acids for expression in a host cell. In addition, theCD27 monoclonal antibodies of the invention define at least threenon-overlapping epitopes on the extracellular domain of CD27 which whenengaged by an antibody of the invention, are prevented from CD70-typeligand ligation driven signaling and downstream biological activity.

Another aspect of the invention is an isolated anti-CD27 antibodyreactive with a CD27 protein epitope defined by residues betweenpositions 21-191 of the CD27 protein.

Another aspect of the invention is an isolated antibody having a heavychain variable region sequence selected from the sequences shown in SEQID NOs: 76, 78, 80, 102-126, 128-136, and 145-147, and a light chainvariable region sequence selected from the sequences shown in SEQ IDNOs: 77, 79, 81-101, 127, 137-144, and 148, including variants of thosesequences, e.g., conservative substitutions.

A further aspect of the invention is an isolated antibody having heavyand light chain CDR sequences selected from the sequences shown in SEQID NOs: 1-75 and 151-158, including variants of those sequences, e.g.,conservative substitutions.

Another aspect of the invention is an isolated polynucleotide encodingan antibody of the invention.

In another aspect, the invention relates to an antibody which binds to acommon epitope defined by the region on the protein to which antibodiesC2177 and/or C2186 or human antibodies generated therefrom described inTables 30-39 bind or which compete for binding to the CD27 protein withantibodies C2177 and/or C2186 or human antibodies generated therefromdescribed in Tables 30-39. In another embodiment, the invention relatesto an antibody which binds to an epitope of the extracellular domain ofCD27 defined by the region on the protein to which antibody C2191 bindor human antibodies generated therefrom described in Tables 30-39 andcompetes for binding to the CD27 protein with antibody C2191 or humanantibodies generated therefrom described in Tables 30-39. In anotherembodiment, invention relates to an antibody which binds to an epitopeof the extracellular domain of CD27 defined by the region on the proteinto which antibody C2192 binds or human antibodies generated therefromdescribed in Tables 30-39 and competes for binding to the CD27 proteinwith antibody C2192 or human antibodies generated therefrom described inTables 30-39. In another aspect, the invention comprises an antibody orfragment thereof derived from one or more of antibodies C2177, C2186,C2191, and C2192 or human antibodies generated therefrom described inTables 30-39 having other functional binding characteristics exhibitedby one or more of antibodies C2177, C2186, C2191, and C2192, or humanantibodies generated therefrom described in Tables 30-39, such asinhibiting the binding of CD27 to CD70 positive cells.

Thus, one aspect of the invention relates to an engineered antibodycomprising an engineered (e.g., humanized or human adapted) heavy chainand light chain, wherein:

-   -   (1) the engineered heavy chain variable region comprises or is        derived from one or more complementarity determining regions        (CDRS) from the mouse antibodies C2177, C2191, C2192 and C2186        heavy chain and a framework from a human acceptor antibody heavy        chain, optionally having one or more human framework residue        substitutions, and    -   (2) the engineered light chain variable region comprises one or        more complementarity determining regions from the mouse        antibodies C2177, C2191, C2192 and C2186 light chain and a        framework from a human acceptor antibody light chain optionally        having one or more human framework residue substitutions; and    -   (3) the engineered antibody specifically binds to human CD27 and        interferes with its interaction with CD70.

In a further embodiment, the engineered antibody may be composed of oneor more CDRs that are further engineered with one or more substitutionsor deletions, for example, those that are 90%, 95%, 98% or 99.5%identical to one or more CDRs of antibodies C2177, C2191, C2192 and/orC2186.

Another embodiment relates to the treatment or prevention ofpathological conditions associated with CD27 bioactivity byadministering a therapeutically or prophylactically effective amount ofan antibody of the present invention, portion thereof or a mixture ofantibodies of the present invention or portions thereof to a subject inneed of such treatment.

In a further embodiment, the invention comprises antigen epitopes as acomponent of a vaccine. The polypeptides or polynucleotides encoding thepolypeptide epitopes described above comprising subfragments orthree-dimensional analogs of some or all of SEQ ID NO: 1 residues21-191, or conservative changes thereof, are recognized by theantibodies of the invention. The polypeptides and polynucleotides areuseful for actively immunizing a host to elicit production of antibodiesagainst CD27 capable of the combating or preventing pathologicalconditions associated with CD27 bioactivity.

The invention also relates to methods of generating, purifying,formulating, and packaging an antibody of the invention for use in thetreatment or prevention of pathological conditions associated with CD27bioactivity by administering a therapeutically or prophylacticallyeffective amount of an antibody or portion thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effects of the C2177 antibody on T-cellproliferation.

FIG. 2 shows the crystal structure of CD27:C2177:C2191 ternary complex.N- and C-termini of CD27 fragment are labeled. Heavy chains of Fabs aredarker than their light chains.

FIG. 3 is a diagram of CD27 protein-C2177 antibody contacts with CD27protein residues (epitope) in circles and C2177 antibody residues(paratope) in boxes.

FIG. 4 is a diagram of CD27 protein-C2191 antibody contacts with CD27protein residues (epitope) are in circles and C2191 antibody residues(paratope) in boxes.

DETAILED DESCRIPTION Abbreviations

CDR—complementarity determining region; CFSE—carboxyfluoresceindiacetate, succinimidyl ester; ECD—extracellular domain; FR—framework;H—heavy chain; GvHD graft-versus-host disease; L—light chain;IFN—interferon (g, gamma); Ig—immunoglobulin; Mab—monoclonal antibody;MMP—matrix metalloproteinase; PBMC—peripheral blood mononuclear cells;VL—Variable light chain; VH—Variable heavy chain

Definitions

As used herein, an “antibody” includes whole antibodies and any antigenbinding fragment or a single chain thereof. Thus, the antibody includesany protein or peptide containing molecule that comprises at least aportion of an immunoglobulin molecule, such as but not limited to, atleast one complementarity determining region (CDR) of a heavy or lightchain or a ligand binding portion thereof, a heavy chain or light chainvariable region, a heavy chain or light chain constant region, aframework (FR) region, or any portion thereof, or at least one portionof a binding protein, which can be incorporated into an antibody of thepresent invention. The term “antibody” is further intended to encompassantibodies, digestion fragments, specified portions and variantsthereof, including antibody mimetics or comprising portions ofantibodies that mimic the structure and/or function of an antibody or aspecified fragment or portion thereof, including single chain and singledomain antibodies and fragments thereof. Functional fragments includeantigen-binding fragments to a preselected target. Examples of bindingfragments encompassed within the term “antigen binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH, domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH,domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, VL and VH, are coded for by separate genes,they can be joined, using recombinant methods, by a synthetic linkerthat enables them to be made as a single protein chain in which the VLand VH regions pair to form monovalent molecules (known as single chainFv (scFv); see e.g., Bird et al. (I 988) Science 242:423-426, and Hustonet al. (1988) Proc. Natl. Acad Sci. USA 85:5879-5883). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. These antibody fragments areobtained using conventional techniques known to those with skill in theart, and the fragments are screened for utility in the same manner asare intact antibodies. Conversely, libraries of scFv constructs can beused to screen for antigen binding capability and then, usingconventional techniques, spliced to other DNA encoding human germlinegene sequences. One example of such a library is the “HuCAL: HumanCombinatorial Antibody Library” (Knappik, A. et al. J Mol Biol (2000)296(1):57-86).

The term “CDR” refers to the complementarity determining region orhypervariable region amino acid residues of an antibody that participatein or are responsible for antigen-binding. The hypervariable region orCDRs of the human IgG subtype of antibody comprise amino acid residuesfrom residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chainvariable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavychain variable domain as described by Kabat et al. (1991 Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md.) and/or those residues froma hypervariable loop (i.e., residues 26-32 (L1), 50-52 (L2) and 91-96(L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2) orthe current H2 Chothia definition of 52-57, and 96-101 (H3) in the heavychain variable domain as described by (Chothia et al., J. Mol. Biol.196: 901-917 (1987)).

Framework or FR1-4 residues are those variable domain residues otherthan and bracketing the hypervariable regions. More recently, auniversal numbering system has been developed and widely adopted,international ImMunoGeneTics information System® (IMGT) (LaFranc, et al.2005. Nucl Acids Res. 33:D593-D597).

Herein, the CDRs are referred to in terms of both the amino acidsequence and the location within the light or heavy chain by sequentialnumbering. As the “location” of the CDRs within the structure of theimmunoglobulin variable domain is conserved between species and presentin structures called loops, by using numbering systems that alignvariable domain sequences according to structural features, CDR andframework residues and are readily identified. This information is usedin grafting and replacement of CDR residues from immunoglobulins of onespecies into an acceptor framework from, typically, a human antibody.

The term “CD27” refers to the human TNF receptor superfamily (TNFSF27),the product of the human gene 939 (CD27 gene), also called human CD27Lreceptor, MGC20393, S152, T14, T-cell activation antigen CD27 andinclude all of the variants, isoforms and species homologs of CD27. Theexpressed human CD27 (NCBI Accession No. NP 001233) is a polypeptide of260 amino acids in length having a 20 amino acid secretion signal at theN-terminus. Accordingly, the antibodies of the invention may, in certaincases, cross-react with CD27 from species other than human. In othercases, the antibodies may be completely specific for human CD27 and notexhibit species or other types of cross-reactivity. By CD27 biologicalactivities is meant, any downstream activities resulting from CD27receptor binding and/or activation as a result of activation of CD27 byone or more ligands, especially CD70 polypeptides (TNFSF7, NP_001243),or other ligands, such as SIVA. CD27 transduces signals that lead to theactivation of NF-κ-β and MAPK8/JNK. Adaptor proteins TRAF2 and TRAF5have been shown to mediate the signaling process of this receptor. CD27biologic activities may also result from the binding of certaintruncated forms of CD27 or fragments of CD27 to ligands which themselvesexhibit biologic activities, for example, a polypeptide which comprisesfrom about residues 21-191 of the full-length protein can bind to CD70.The CD27 antigen cytoplasmic tail, residues 213-260, binds to theN-terminus of the SIVA protein (also known as the apoptosis-inducingfactor: CD27BP; SIVA1, Siva-1, NP_006418 (175 aa)); and Siva-2, SIVA2,NP_068355 (110 aa).

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three-dimensional structural characteristics,as well as specific charge characteristics. Conformational andnon-conformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

“Humanization” (also called Reshaping or CDR-grafting) or “engineering”includes established techniques for reducing the immunogenicity ofmonoclonal antibodies (mAbs) from xenogeneic sources (commonly rodent)and for improving affinity or the effector functions (ADCC, complementactivation, C1q binding). The engineered mAb can be produced using thetechniques of molecular biology, using phage displayed randomizedsequences, or synthesized de novo. For example, in order to construct ahumanized antibody with incorporated CDR regions from a nonhumanspecies, the design might include variations, such as conservative aminoacid substitutions in residues of the CDRs, and back substitution ofresidues from the nonhuman mAb into the human framework regions(backmutations). The positions can be discerned or identified bysequence comparison methods, consensus sequence analysis, or structuralanalysis of the variable regions' 3D structure. Computer programs areavailable which illustrate and display probable three-dimensionalconformational structures of selected candidate immunoglobulinsequences. Inspection of these displays permits analysis of the likelyrole of the residues in the functioning of the candidate immunoglobulinsequence, i.e., the analysis of residues that influence the ability ofthe candidate immunoglobulin to bind its antigen. In this way or bysimple sequence alignment algorithms (e.g., Clustal W), FR (framework)residues can be selected from known antibody sequences, found in suchpublicly accessible databases as VBASE or Kabat, and the consensussequences optimized so that the desired antibody characteristic, such asaffinity for the target antigen(s), is achieved. As the datasets ofknown parameters for antibody structures increases, so does thesophistication and refinement of these techniques. Another approach tohumanization is to modify only surface residues of the rodent sequencewith the most common residues found in human mAbs and has been termed“resurfacing” or “veneering.” A large number of both human and non-humanIg sequences are now known and freely available and used by thoseskilled in the art, e.g., the database and tools developed by LeFranc etal found under the name IMGT; websites curated by the U.S. NationalCenter for Biologics (NCBI); Kabat et al., Sequences of Proteins ofImmunological Interest, U.S. Dept. Health (1983) now also greatlyexpanded and available online, each entirely incorporated herein byreference. Humanization or engineering of antibodies of the presentinvention can be performed using any method known or those developedusing human immunoglobulin sequence information. Such methods are taughtin, for example, Winter U.S. Pat. No. 6,982,361 and Bowdish et al.WO03/025019, the contents of which are incorporated herein by reference.

As used herein, K_(D) refers to the dissociation constant, specifically,the antibody K_(D) for a predetermined antigen, and is a measure ofaffinity of the antibody for a specific target. High affinity antibodieshave a K_(D) of 10⁻⁸M or less, more preferably 10⁻⁹ M or less and evenmore preferably 10⁻¹⁰ M or less, for a predetermined antigen. Thereciprocal of K_(D) is K_(A), the association constant. The term“k_(dis)” or “k₂,” or “k_(d)” as used herein, is intended to refer tothe dissociation rate of a particular antibody-antigen interaction. The“K_(D)” is the ratio of the rate of dissociation (k₂), also called the“off-rate (k_(off))” to the rate of association rate (k₁) or “on-rate(k_(on)).” Thus, K_(D) equals k₂/k₁ or k_(off)/k_(on), and is expressedas a molar concentration (M). It follows that the smaller the K_(D), thestronger the binding. Thus, a K_(D) of 10⁻⁶ M (or 1 microM) indicatesweak binding compared to 10⁻⁹ M (or 1 nM). These values may becalculated using surface plasmon resonance and/or the Kinexa method asknown in the art.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope. Theterm also includes “recombinant antibody” and “recombinant monoclonalantibody” as all antibodies are prepared, expressed, created or isolatedby recombinant means, such as (a) antibodies isolated from an animal ora hybridoma prepared by the fusion of antibody secreting animal cellsand an fusion partner, (b) antibodies isolated from a host celltransformed to express the antibody, e.g., from a transfectoma, (c)antibodies isolated from a recombinant, combinatorial human or otherspecies antibody library, and (d) antibodies prepared, expressed,created or isolated by any other means that involve splicing ofimmunoglobulin gene sequences to other DNA sequences. An “isolatedantibody,” as used herein, is intended to refer to an antibody which issubstantially free of other antibodies having different antigenicspecificities. An isolated antibody that specifically binds to anepitope, isoform or variant of human CD27 may, however, havecross-reactivity to other related antigens, e.g., from other species(e.g., CD27 species homologs). Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals. In oneembodiment of the invention, a combination of “isolated” monoclonalantibodies having different specificities are combined in a well definedcomposition.

As used herein, “specific binding,” “immunospecific binding” and “bindsimmunospecifically” refers to antibody binding to a predeterminedantigen. Typically, the antibody binds with a dissociation constant(K_(D)) of 10⁻⁷ M or less, and binds to the predetermined antigen with aK_(D) that is at least twofold less than its K_(D) for binding to anon-specific antigen (e.g., BSA, casein, or any other specifiedpolypeptide) other than the predetermined antigen. The phrases “anantibody recognizing an antigen” and “an antibody specific for anantigen” are used interchangeably herein with the term “an antibodywhich binds specifically to an antigen.” As used herein “highlyspecific” binding means that the relative K_(D) of the antibody for thespecific target epitope is at least 10-fold less than the K_(D) forbinding that antibody to other ligands.

As used herein, “isotype” refers to the antibody class (e.g., IgM orIgG) that is encoded by heavy chain constant region genes. Some antibodyclasses further encompass subclasses which are also encoded by the heavychain constant regions and further decorated by oligosaccharides atspecific residues within the constant region domains (e.g. IgG1, IgG2,IgG3 and IgG4) which further impart biological functions to theantibody. For example, in human antibody isotypes IgG1, IgG3 and to alesser extent, IgG2 display effector functions as do murine IgG2aantibodies.

By “effector” functions or “effector positive” is meant that theantibody comprises domains distinct from the antigen specific bindingdomains capable of interacting with receptors or other blood componentssuch as complement, leading to, for example, the recruitment ofmacrophages and events leading to destruction of cells bound by theantigen binding domains of the antibody. Antibodies have severaleffector functions mediated by binding of effector molecules. Forexample, binding of the C1 component of complement to antibodiesactivates the complement system. Activation of complement is importantin the opsonisation and lysis of cell pathogens. The activation ofcomplement stimulates the inflammatory response and may also be involvedin autoimmune hypersensitivity. Further, antibodies bind to cells viathe Fc region, with a Fc receptor site on the antibody Fc region bindingto a Fc receptor (FcR) on a cell. There are a number of Fc receptorswhich are specific for different classes of antibody, including IgG(gamma receptors), IgE (eta receptors), IgA (alpha receptors) and IgM(mu receptors). Binding of antibody to Fc receptors on cell surfacestriggers a number of important and diverse biological responsesincluding engulfment and destruction of antibody-coated particles,clearance of immune complexes, lysis of antibody-coated target cells bykiller cells (called antibody-dependent cell-mediated cytotoxicity, orADCC), release of inflammatory mediators, placental transfer and controlof immunoglobulin production.

1. Composition of an Antibody of the Invention

A CD27-neutralizing antibody of the invention is an antibody thatinhibits, blocks, or interferes with at least one CD27 activity or CD70binding, in vitro, in situ and/or in vivo and does not promote,stimulate, induce, or agonize CD27 activity or ligand binding nor doesantibody binding mimic the downstream effects of CD27-ligand ligation,in particular CD70 interaction with CD27, such as signal transduction ina host cell. A suitable CD27-neutralizing antibody, specified portion,or variant can also, optionally, affect at least one CD27 activity orfunction, such as but not limited to, RNA, DNA or protein synthesis,protein release, T-cell activation, B-cell proliferation ordifferentiation, antibody secretion, CD27 receptor signaling, CD27cleavage, CD27-ligand binding, CD27 or CD70 induction, synthesis orsecretion.

In relation to the CD27:CD70 co-stimulatory pathway blocking activity ofthe CD27-neutralizing antibodies of the present invention, the treatmentof autoimmune disorders with elevated T- or B-cell effector functionsmay be beneficial.

The present invention is based upon the discovery of anti-human CD27monoclonal antibodies capable of inhibiting CD27 activation by CD70 andincapable of CD27 self-activation in the absence of CD70 stimulus.Hybridomas and transfectomas capable of secreting such an antibody weregenerated. An NF-kβ reporter gene assay was used to identify severalcandidate antibodies capable of inhibiting CD70-mediated NF-kβ reporteractivation of CD27 expressing host cells. Second, the antibodies werecharacterized as being unable to induce dose-dependent agonisticactivity when incubated with CD27 coupled luciferase reportertransfected cells in the absence of CD70 stimulus. Third, it wasdemonstrated that the antibodies dose-dependently inhibit CD70-dependenthuman naïve CD4 T-cell proliferation. Fourth, the CD27-neutralizingantibodies generated are capable of reducing CD70-mediated stimulationof plasma cell generation from human primary B-cells in a dose dependentmanner. Fifth, no significant dose-dependent agonistic activity wasobserved in primary T- or B-cells with tested anti-CD27 antibodies.

The antibodies of the invention can interfere with CD27:CD70 ligation,inhibit both T-cell effector functions and B-cell differentiation toplasma cells in cell culture and thus may be beneficial for treatment ofimmune-mediated diseases including, but not limited to, rheumatoidarthritis, systemic lupus erythematosus, multiple sclerosis,inflammatory bowel disease, Crohn's Disease, chronic obstructivepulmonary disease or other syndrome, pathology, disease or disorderrelated to the aberrant functions or activation of CD27-expressing cellpopulations. The CD27-binding antibodies described herein recognize atleast three distinct regions on the extracellular domain of human CD27,indicating the additional discovery of multiple sites on CD27 suitablefor the targeting of antibodies or other compounds with similar functionblocking capabilities. Thus, expression and purification of the antibodybinding domains provided herein as amino acid sequences further providesa tool which can be the means for selection of novel moleculesexhibiting CD27-neutralizing activity.

In one embodiment, the anti-human CD27 antibody, has a binding regioncomprising a light chain variable (VL) or heavy chain variable (VH)region having the amino acid sequence as shown in SEQ ID NO: 76-144 andwhich antibody or binding portion thereof immunospecifically binds CD27.In another embodiment of the invention, the antibody or antigen bindingportion thereof, binds to CD27 protein and, in addition, the antibodiespossesses specified functional properties of antibodies of theinvention, such as:

binding to immobilized human CD27;

inhibition of human soluble CD27 binding to cells expressing CD70;

inhibition of human CD70 mediated CD27 signaling measured by NF-kappaBreporter gene assay at an IC50 of less than 0.5 ug/ml;

inhibition of CD70-mediated proliferation of naïve T-cells;

inhibition of CD70 mediated plasma blast formation from primary humanB-cells;

inhibition of human CD70-mediated soluble mediator release from T and Bprimary cells or cell lines;

binding to human CD27 with K_(d) of less than 100 nM (10⁻⁷ M);

minimal activation of CD27 signaling in the absence of CD70 stimulus;and

binding to an epitope on the human CD27 extracellular domain to whichthe Mabs having one or more of the variable region sequences of SEQ IDNOS: 76-144 bind and competes for binding with the Mabs identifiedhaving one or more of the variable region sequences of SEQ ID NOS:76-144.

Since it is well known in the art that antibody heavy and light chainsCDR domains play a particularly important role in the bindingspecificity/affinity of an antibody for an antigen, the recombinantantibodies of the invention disclosed herein preferably comprise one ormore of the heavy and light chain CDRs of SEQ ID NOS: 1-75. Suchantibodies can be prepared by chemically joining together the variousportions (e.g., CDRs, framework) of the antibody using conventionaltechniques, by preparing and expressing a (i.e., one or more) nucleicacid molecule that encodes the antibody using conventional techniques ofrecombinant DNA technology or by using any other suitable method.

In one embodiment, the human antibodies of the invention have thesequence of one or more of the heavy and light chain CDRs of SEQ ID NOS:1-75. In addition to these CDR sequences, the ordinarily skilled artisanwill appreciate that some deviation from the exact CDR sequences may bepossible or desirable while still retaining the ability of the antibodyto bind CD27 (e.g., conservative substitutions). Accordingly, in anotherembodiment, the human antibody may be composed of one or more CDRs thatare, for example, 90%, 95%, 98% or 99.5% identical to the CDRs listed inSEQ ID NOs: 1-75.

In another embodiment, the epitope bound by the antibodies of theinvention, comprising as few as five to all of residues 21-191 of CD27protein or a nucleic acid coding sequence therefor, can be used toimmunize a subject in order to produce the antibodies of the inventiondirectly in the host for the purpose of treating, preventing, orameliorating disease or symptoms of disease associated with theproduction of CD27.

2. Generation of CD27-Neutralizing Antibodies

A CD27-neutralizing antibody exhibiting the desired bioactivity spectrumas exemplified herein by the disclosed and described antibodies, can begenerated by a variety of techniques, including the standard somaticcell hybridization technique (hybridoma method) of Kohler and Milstein(1975) Nature 256:495. In the hybridoma method, a mouse or otherappropriate host animal, such as a hamster or macaque monkey, isimmunized as described herein to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization. Alternatively, lymphocytes may beimmunized in vitro. Lymphocytes then are fused with myeloma cells usinga suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice,pp. 59-103 (Academic Press, 1986)).

A CD27-neutralizing antibody can also be optionally generated byimmunization of a transgenic animal (e.g., mouse, rat, hamster,non-human primate, and the like) capable of producing a repertoire ofhuman antibodies, as described herein and/or as known in the art. Cellsthat produce a human anti-CD27 antibody can be isolated from suchanimals and immortalized using suitable methods, such as the methodsdescribed herein. Alternatively, the antibody coding sequences may becloned, introduced into a suitable vector, and used to transfect a hostcell for expression and isolation of the antibody by methods taughtherein and those known in the art.

The use of transgenic mice carrying human immunoglobulin (Ig) loci intheir germline configuration provides for the isolation of high affinityfully human monoclonal antibodies directed against a variety of targetsincluding human self antigens for which the normal human immune systemis tolerant (Lonberg, N. et al., U.S. Pat. Nos. 5,569,825, 6,300,129 and1994, Nature 368:856-9; Green, L. et al., 1994, Nature Genet. 7:13-21;Green, L. & Jakobovits, 1998, Exp. Med. 188:483-95; Lonberg, N andHuszar, D., 1995, Int. Rev. Immunol. 13:65-93; Kucherlapati, et al. U.S.Pat. No. 6,713,610; Bruggemann, M. et al., 1991, Eur. J. Immunol.21:1323-1326; Fishwild, D. et al., 1996, Nat. Biotechnol. 14:845-851;Mendez, M. et al., 1997, Nat. Genet. 15:146-156; Green, L., 1999, J.Immunol. Methods 231:11-23; Yang, X. et al., 1999, Cancer Res.59:1236-1243; Brüggemann, M. and Taussig, M J., Curr. Opin. Biotechnol.8:455-458, 1997; Tomizuka et al. WO02043478). The endogenousimmunoglobulin loci in such mice can be disrupted or deleted toeliminate the capacity of the animal to produce antibodies encoded byendogenous genes. In addition, companies, such as Abgenix, Inc.(Freemont, Calif.) and Medarex (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology as described above.

In another embodiment, the human antibody is selected from a phagelibrary, where that phage comprises human immunoglobulin genes and thelibrary expresses human antibody binding domains as, for example, singlechain antibodies (scFv), as Fabs, or some other construct exhibitingpaired or unpaired antibody variable regions (Vaughan et lo al. NatureBiotechnology 14:309-314 (1996): Sheets et al. PITAS (USA) 95:6157-6162(1998)); Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks etal. J. Mol. Biol., 222:581 (1991)). Human monoclonal antibodies of theinvention can also be prepared using phage display methods for screeninglibraries of human immunoglobulin genes. Such phage display methods forisolating human antibodies are established in the art. See for example:U.S. Pat. Nos. 5,223,409; 5,403,484; and U.S. Pat. No. 5,571,698 toLadner et al.; U.S. Pat. Nos. 5,427,908 and 5, 580,717 to Dower et al.;U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S.Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and6,593,081 to Griffiths et al.

Preparation of immunogenic antigens, and monoclonal antibody productioncan be performed using any suitable technique, such as recombinantprotein production. The immunogenic antigens can be administered to ananimal in the form of purified protein, or protein mixtures includingwhole cells or cell or tissue extracts, or the antigen can be formed denovo in the animal's body from nucleic acids encoding said antigen or aportion thereof.

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, (c) purificationtechniques, or combinations thereof, as well-known in the art. DNAencoding the monoclonal antibodies is readily isolated and sequencedusing methods known in the art (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies). Where a hybridoma is produced, suchcells can serve as a source of such DNA. Alternatively, using displaytechniques wherein the coding sequence and the translation product arelinked, such as phage or ribosomal display libraries, the selection ofthe binder and the nucleic acid is simplified. After phage selection,the antibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria.

Humanized Antibodies

The invention further provides humanized (engineered or human adapted)immunoglobulins (or antibodies) which bind human CD27. The humanizedforms of immunoglobulins have variable framework region(s) substantiallyfrom a human immunoglobulin (termed an acceptor immunoglobulin) and CDRssubstantially from a non-human Mab which specifically binds CD27. Theconstant region(s), if present, are also substantially from a humanimmunoglobulin. The humanized antibodies exhibit K_(D) for CD27 of atleast about 10⁻⁶ M (1 microM), about 10⁻⁷ M (100 nM), or less. Thebinding affinity of the humanized antibodies may be greater or less thanthat of the mouse antibody from which they were derived. To affect achange in affinity, e.g., improve affinity, of the humanized antibodyfor CD27 substitutions in either the CDR residues or the human residuesmay be made.

The substitution of mouse CDRs into a human variable domain framework ismost likely to result in retention of their correct spatial orientationif the human variable domain framework adopts the same or similarconformation to the mouse variable framework from which the CDRsoriginated. This is achieved by obtaining the human variable domainsfrom human antibodies whose framework sequences exhibit a high degree ofsequence identity with the murine variable framework domains from whichthe CDRs were derived. The heavy and light chain variable frameworkregions can be derived from the same or different human antibodysequences. The human antibody sequences can be the sequences ofnaturally occurring human antibodies, be derived from human germlineimmunoglobulin sequences, or can be consensus sequences of several humanantibody and/or germline sequences.

Suitable human antibody sequences are identified by computer comparisonsof the amino acid sequences of the mouse variable regions with thesequences of known human antibodies. The comparison is performedseparately for heavy and light chains but the principles are similar foreach.

In one example, the amino acid sequence of a CD27-neutralizing mAb isused to query a human antibody database compiled from public antibodysequence databases. The heavy chain variable regions disclosed ordescribed herein can be used to find the human variable region with thehighest sequence identity. The variable region of the light chaindisclosed or described herein can, similarly, be used to find the humanvariable region with the highest sequence identity. A DNA construct inwhich the regions coding for the CDRs of one of the heavy chain variableregions from the murine Mab donor are transferred into the selectedhuman heavy chain variable sequence, replacing the CDRs of the humanvariable region is prepared for each murine variable region.

The unnatural juxtaposition of murine CDR regions with human variableframework region can result in unnatural conformational restraints,which, unless corrected by substitution of certain amino acid residues,lead to loss of binding affinity.

As noted supra, the humanized antibodies of the invention comprisevariable framework region(s) substantially from a human immunoglobulinand CDRs substantially from a mouse immunoglobulin (e.g., C2177, C2186,C2191, or C2192 mouse antibodies). Having identified the CDRs of mouseantibodies and appropriate human acceptor immunoglobulin sequences, thenext step is to determine which, if any, residues from these componentsshould be substituted to optimize the properties of the resultinghumanized antibody. In general, substitution of human amino acidresidues with murine should be minimized, because introduction of murineresidues increases the risk of the antibody eliciting a HAMA response inhumans. Amino acids are selected for substitution based on theirpossible influence on CDR conformation and/or binding to antigen.Investigation of such possible influences can be done by modeling,examination of the characteristics of the amino acids at particularlocations, or empirical observation of the effects of substitution ormutagenesis of particular amino acids. With regard to the empiricalmethod, it has been found to be particularly convenient to create alibrary of variant sequences that can be screened for the desiredactivity, binding affinity or specificity. One format for creation ofsuch a library of variants is a phage display vector. Alternatively,variants can be generated using other methods for varigation of anucleic acid sequence encoding the targeted residues within the variabledomain.

Another method of determining whether further substitutions arerequired, and the selection of amino acid residues for substitution, canbe accomplished using computer modeling. Computer hardware and softwarefor producing three-dimensional images of immunoglobulin molecules arewidely available. In general, molecular models are produced startingfrom solved structures for immunoglobulin chains or domains thereof. Thechains to be modeled are compared for amino acid sequence similaritywith chains or domains of solved three dimensional structures, and thechains or domains showing the greatest sequence similarity is/areselected as starting points for construction of the molecular model. Thesolved starting structures are modified to allow for differences betweenthe actual amino acids in the immunoglobulin chains or domains beingmodeled, and those in the starting structure. The modified structuresare then assembled into a composite immunoglobulin. Finally, the modelis refined by energy minimization and by verifying that all atoms arewithin appropriate distances from one another and that bond lengths andangles are within chemically acceptable limits.

Usually the CDR regions in humanized antibodies are substantiallyidentical, and more usually, identical to the corresponding CDR regionsin the mouse antibody from which they were derived. Although not usuallydesirable, it is sometimes possible to make one or more conservativeamino acid substitutions of CDR residues without appreciably affectingthe binding affinity of the resulting humanized immunoglobulin.Occasionally, substitutions of CDR regions can enhance binding affinity.

Other than for the specific amino acid substitutions discussed above,the framework regions of humanized immunoglobulins are usuallysubstantially identical, and, more usually, identical to the frameworkregions of the human antibodies from which they were derived. Of course,many of the amino acids in the framework region make little or no directcontribution to the specificity or affinity of an antibody. Thus, manyindividual conservative substitutions of framework residues can betolerated without appreciable change of the specificity or affinity ofthe resulting humanized immunoglobulin.

Because of the degeneracy of the code, a variety of nucleic acidsequences will encode each immunoglobulin amino acid sequence. Thedesired nucleic acid sequences can be produced by de nova solid-phaseDNA synthesis or by PCR mutagenesis of an earlier prepared variant ofthe desired polynucleotide. All nucleic acids encoding the antibodiesdescribed in this application are expressly included in the invention.

The variable segments of humanized antibodies produced as describedsupra are typically linked to at least a portion of a humanimmunoglobulin constant region. The antibody will contain both lightchain and heavy chain constant regions. The heavy chain constant regionusually includes CH1, hinge, CH2, CH3, and, sometimes, CH4 domains.

The humanized antibodies may comprise any type of constant domains fromany class of antibody, including IgM, IgG, IgD, IgA and IgE, and anysubclass (isotype), including IgG1, IgG2, IgG3 and IgG4. When it isdesired that the humanized antibody exhibit cytotoxic activity, theconstant domain is usually a complement-fixing constant domain and theclass is typically IgG₁. When such cytotoxic activity is not desirable,the constant domain may be of the IgG₂ class. The humanized antibody maycomprise sequences from more than one class or isotype.

Nucleic acids encoding humanized light and heavy chain variable regions,optionally linked to constant regions, are inserted into expressionvectors. The light and heavy chains can be cloned in the same ordifferent expression vectors. The DNA segments encoding immunoglobulinchains are operably linked to control sequences in the expressionvector(s) that ensure the expression of immunoglobulin polypeptides.Such control sequences include a signal sequence, a promoter, anenhancer, and a transcription termination sequence (see Queen et al.,Proc. Natl. Acad. Sci. USA 86, 10029 (1989); WO 90/07861; Co et al., J.Immunol. 148, 1149 (1992), which are incorporated herein by reference intheir entirety for all purposes).

Efficacy of a therapeutic protein can be limited by unwanted immunereactions. Non-human monoclonal antibodies can have substantialstretches of linear amino acid sequences and local structuralconformations that can elicit immune response in humans. The firstattempt to reduce immunogenicity of non-human antibodies was theconstruction of human-murine antibody chimeras, which was then followedby methods for humanization of those chimeras in the late 1980's (reviewin Almagro and Fransson, Front Biosci 13: 1619-1633, 2008).

One of the most often used humanization approaches is the so-called“Complementarity-Determining Regions (CDR) grafting” wherein murineCDR's are grafted into human antibody Framework Regions (FR's).Nevertheless, application of this method more often than not results ina substantial loss of binding to antigen and thus a reduction in potencyof the antibody-based drug. Hence, it is highly valuable to use sounddesign principles for creating antibody molecules that elicit minimalimmunogenic reactions while retaining the binding and biophysicalprofiles of the parent non-human molecule when injected into humans.

The humanization of 2177 and 2191, two mouse monoclonal antibodies (mAb)with binding specificity to CD27 is described. The frameworks (FR) ofthese antibodies were replaced by human germline gene FRs using thefirst step of the Janssen proprietary humanization technology calledHuman Framework Adaption (HFA) disclosed in the patent applicationRaghunathan, G., US20090118127 A1 and further exemplified in Fransson etal (J Mol Biol 398:214-231, 2010). This technology enables a set of mAbsspecific for CD27 with superior binding and inhibition properties tothose measured for the parental mouse antibodies 2177 and 2191.

3. Methods of Using an Anti-CD27 Antibody

As described in detail below, the present invention demonstrates thatfour isolated monoclonal antibodies (C2177, C2186, C2191, and C2192)bind three non-overlapping epitopes on CD27 and display in vitro and/orin vivo CD27 inhibiting activities. Significantly, the reactivity of theMAbs includes the ability to dose-dependently block CD27 interactionwith CD70, reduce CD27 signaling in the presence of CD70, reduce IL-4and IFNg production by T-cells, and inhibit CD70-dependent human naïveCD4+ T-cell proliferation, CD70-dependent B-cell proliferation andplasma cell generation. Moreover, isolated antibodies do notsignificantly induce CD27 activation in the absence of CD70 stimulus.

Given the properties of the monoclonal antibodies as described in thepresent invention, the antibodies or antigen binding fragments thereofare suitable both as therapeutic and prophylactic agents for treating orpreventing CD27-associated conditions in humans and animals.

In general, use will comprise administering a therapeutically orprophylactically effective amount of one or more monoclonal antibodiesor antigen binding fragments of the present invention, or an antibody ormolecule selected to have similar spectra of binding and biologicactivity, to a susceptible subject or one exhibiting a condition inwhich CD27 activity is known to have pathological sequelae, such asimmunological disorders or tumor growth and metastasis. Any active formof the antibody can be administered, including Fab and F(ab′)2fragments.

Preferably, the antibodies used are compatible with the recipientspecies such that the immune response to the MAbs does not result in anunacceptably short circulating half-life or induce an immune response tothe MAbs in the subject. The MAbs administered may exhibit somesecondary functions, such as binding to Fc receptors of the subject andactivation of ADCC mechanisms, in order to deplete the target cellpopulation using cytolytic or cytotoxic mechanisms or they may beengineered to by limited or devoid of these secondary effector functionsin order to preserve the target cell population.

Treatment of individuals may comprise the administration of atherapeutically effective amount of the antibodies of the presentinvention. The antibodies can be provided in a kit as described below.The antibodies can be used or administered as a mixture, for example, inequal amounts, or individually, provided in sequence, or administeredall at once. In providing a patient with antibodies, or fragmentsthereof, capable of binding to CD27, or an antibody capable ofprotecting against CD27 in a recipient patient, the dosage ofadministered agent will vary depending upon such factors as thepatient's age, weight, height, sex, general medical condition, previousmedical history, etc.

In a similar approach, another therapeutic use of the monoclonalantibodies of the present invention is the active immunization of apatient using an anti-idiotypic antibody raised against one of thepresent monoclonal antibodies. Immunization with an anti-idiotype whichmimics the structure of the epitope could elicit an active anti-CD27response (Linthicum, D. S. and Farid, N. R., Anti-idiotypes, Receptors,and Molecular Mimicry (1988), pp 1-5 and 285-300).

Likewise, active immunization can be induced by administering one ormore antigenic and/or immunogenic epitopes as a component of a vaccine.Vaccination could be performed orally or parenterally in amountssufficient to enable the recipient to generate protective antibodiesagainst this biologically functional region, prophylactically ortherapeutically. The host can be actively immunized with theantigenic/immunogenic peptide in pure form, a fragment of the peptide,or a modified form of the peptide. One or more amino acids, notcorresponding to the original protein sequence can be added to the aminoor carboxyl terminus of the original peptide, or truncated form ofpeptide. Such extra amino acids are useful for coupling the peptide toanother peptide, to a large carrier protein, or to a support. Aminoacids that are useful for these purposes include: tyrosine, lysine,glutamic acid, aspartic acid, cysteine and derivatives thereof.Alternative protein modification techniques may be used, e.g.,NH2-acetylation or COOH-terminal amidation, to provide additional meansfor coupling or fusing the peptide to another protein or peptidemolecule or to a support.

The antibodies capable of protecting against unwanted CD27 bioactivityare intended to be provided to recipient subjects in an amountsufficient to effect a reduction, resolution, or amelioration in theCD27-related symptom or pathology. An amount is said to be sufficient ora “therapeutically effective amount” to “effect” the reduction ofsymptoms if the dosage, route of administration, etc. of the agent aresufficient to influence such a response. Responses to antibodyadministration can be measured by analysis of subject's affectedtissues, organs, or cells as by imaging techniques or by ex vivoanalysis of tissue samples. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient patient.

Therapeutic Applications

The CD27-neutralizing antibodies of the present invention, antigenbinding fragments, or specified variants thereof can be used to measureor cause effects in an cell, tissue, organ or animal (including mammalsand humans), to diagnose, monitor, modulate, treat, alleviate, helpprevent the incidence of, or reduce the symptoms of, a conditionmediated, affected or modulated by CD27 or cells expressing CD27. Thus,the present invention provides a method for modulating or treating atleast one CD27 related disease, in a cell, tissue, organ, animal, orpatient, as known in the art or as described herein, using at least oneCD27 antibody of the present invention. Particular indications arediscussed below.

Immune Related Disease

The present invention also provides a method for modulating or treatingan immune related inflammatory disease, in a cell, tissue, organ,animal, or patient including, but not limited to rheumatoid arthritis,juvenile rheumatoid arthritis, systemic onset juvenile rheumatoidarthritis, psoriatic arthritis, ankylosing spondilitis, gastric ulcer,seronegative arthropathies, osteoarthritis, inflammatory bowel disease,ulcerative colitis, systemic lupus erythematosis, antiphospholipidsyndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonaryfibrosis, systemic vasculitis/wegener's granulomatosis, sarcoidosis,orchitis/vasectomy reversal procedures, allergic/atopic diseases,asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergicconjunctivitis, hypersensitivity pneumonitis, transplants, organtransplant rejection, graft-versus-host disease, systemic inflammatoryresponse syndrome, sepsis syndrome, gram positive sepsis, gram negativesepsis, culture negative sepsis, fungal sepsis, neutropenic fever,urosepsis, meningococcemia, trauma/hemorrhage, burns, ionizing radiationexposure, acute pancreatitis, adult respiratory distress syndrome,rheumatoid arthritis, alcohol-induced hepatitis, chronic inflammatorypathologies, sarcoidosis, Crohn's pathology, sickle cell anemia,diabetes, nephrosis, atopic diseases, hypersensitity reactions, allergicrhinitis, hay fever, perennial rhinitis, conjunctivitis, endometriosis,asthma, urticaria, systemic anaphalaxis, dermatitis, pernicious anemia,hemolytic disesease, thrombocytopenia, graft rejection of any organ ortissue, kidney translplant rejection, heart transplant rejection, livertransplant rejection, pancreas transplant rejection, lung transplantrejection, bone marrow transplant (BMT) rejection, skin allograftrejection, cartilage transplant rejection, bone graft rejection, smallbowel transplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynoud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,and skin changes syndrome), antiphospholipid syndrome, pemphigus,scleroderma, mixed connective tissue disease, idiopathic Addison'sdisease, diabetes mellitus, chronic active hepatitis, primary billiarycirrhosis, vitiligo, vasculitis, post-MI cardiotomy syndrome, type IVhypersensitivity, contact dermatitis, hypersensitivity pneumonitis,allograft rejection, granulomas due to intracellular organisms, drugsensitivity, metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,cachexia, cystic fibrosis, neonatal chronic lung disease, chronicobstructive pulmonary disease (COPD), familial hematophagocyticlymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,nephrotic syndrome, nephritis, glomerular nephritis, acute renalfailure, hemodialysis, uremia, toxicity, preeclampsia, OKT3 therapy,anti-CD3 therapy, cytokine therapy, chemotherapy, radiation therapy(e.g., including but not limited to asthenia, anemia, cachexia, and thelike), chronic salicylate intoxication, and the like.

Pulmonary Disease

The present invention also provides a method for modulating or treatinga pulmonary or pleural disease in a cell, tissue, organ, animal orpatient, including, but not limited to, modulating the immune-responseto associated or ancillary cells or cellular processes involving CD27in, for example, pneumonia; lung abscess; occupational lung diseasescaused be agents in the form or dusts, gases, or mists; asthma,bronchiolitis fibrosa obliterans, respiratory failure, hypersensitivitydiseases of the lungs including hypersensitivity pneumonitis (extrinsicallergic alveolitis), allergic bronchopulmonary aspergillosis, and drugreactions; adult respiratory distress syndrome (ARDS), Goodpasture'sSyndrome, chronic obstructive airway disorders (COPD), idiopathicinterstitial lung diseases such as idiopathic pulmonary fibrosis,sarcoidosis, desquamative interstitial pneumonia, acute interstitialpneumonia, respiratory bronchiolitis-associated interstitial lungdisease, idiopathic bronchiolitis obliterans with organizing pneumonia,lymphocytic interstitial pneumonitis, Langerhans' cell granulomatosis,idiopathic pulmonary hemosiderosis; acute bronchitis, pulmonary alveolarproteinosis, bronchiectasis, pleural disorders, atelectasis, cysticfibrosis, and tumors of the lung, and pulmonary embolism.

Malignant Disease

The present invention also provides a method for modulating or treatinga malignant disease in a cell, tissue, organ, animal or patient,including, but not limited to, modulating the immune-response toassociated or ancillary cells or cellular processes involving CD27 in,at least one of: leukemia, acute leukemia, acute lymphoblastic leukemia(ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chronicmyelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairycell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin'sdisease, a malignamt lymphoma, non-hodgkin's lymphoma, Burkitt'slymphoma, multiple myeloma, solid tumors as primary disease or asmetastatic disease, Kaposi's sarcoma, colorectal carcinoma, pancreaticcarcinoma, renal cell carcinoma, lung cancer including mesothelioma,breast cancer, nasopharyngeal carcinoma, malignant histiocytosis,paraneoplastic syndrome/hypercalcemia of malignancy, adenocarcinomas,squamous cell carcinomas, sarcomas, malignant melanoma, particularlymetastatic melanoma, hemangioma, metastatic disease, cancer related boneresorption, cancer related bone pain, and the like.

Cardiovascular Disease

The present invention also provides a method for modulating or treatinga cardiovascular disease in a cell, tissue, organ, animal, or patient,including, but not limited to, modulating the immune-response toassociated or ancillary cells or cellular processes involving CD27 in,at least one of myocardial infarction, congestive heart failure, stroke,ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis,restenosis, diabetic atheriosclerotic disease, hypertension, arterialhypertension, renovascular hypertension, syncope, shock, syphilis of thecardiovascular system, heart failure, cor pulmonale, primary pulmonaryhypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter,atrial fibrillation (sustained or paroxysmal), post perfusion syndrome,cardiopulmonary bypass inflammation response, chaotic or multifocalatrial tachycardia, regular narrow QRS tachycardia, specific arrythmias,ventricular fibrillation, His bundle arrythmias, atrioventricular block,bundle branch block, myocardial ischemic disorders, coronary arterydisease, angina pectoris, myocardial infarction, cardiomyopathy, dilatedcongestive cardiomyopathy, restrictive cardiomyopathy, valvular heartdiseases, endocarditis, pericardial disease, cardiac tumors, aordic andperipheral aneuryisms, aortic dissection, inflammation of the aorta,occulsion of the abdominal aorta and its branches, peripheral vasculardisorders, occulsive arterial disorders, peripheral atherloscleroticdisease, thromboangitis obliterans, functional peripheral arterialdisorders, Raynaud's phenomenon and disease, acrocyanosis,erythromelalgia, venous diseases, venous thrombosis, varicose veins,arteriovenous fistula, lymphederma, lipedema, unstable angina,reperfusion injury, post pump syndrome, ischemia-reperfusion injury, andthe like.

Neurologic Disease

The present invention also provides a method for modulating or treatingat neurologic disease in a cell, tissue, organ, animal or patient,including, but not limited to, modulating the immune-response toassociated or ancillary cells or cellular processes involving CD27 in:neurodegenerative diseases, multiple sclerosis, migraine headache, AIDSdementia complex, demyelinating diseases, such as multiple sclerosis andacute transverse myelitis; extrapyramidal and cerebellar disorders' suchas lesions of the corticospinal system; disorders of the basal gangliaor cerebellar disorders; hyperkinetic movement disorders, such asHuntington's Chorea and senile chorea; drug-induced movement disorders,such as those induced by drugs which block CNS dopamine receptors;hypokinetic movement disorders, such as Parkinson's disease; Progressivesupranucleo Palsy; structural lesions of the cerebellum; spinocerebellardegenerations, such as spinal ataxia, Friedreich's ataxia, cerebellarcortical degenerations, multiple systems degenerations (Mencel,Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic disorders(Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, andmitochondrial multi.system disorder); demyelinating core disorders, suchas multiple sclerosis, acute transverse myelitis; and disorders of themotor unit, such as neurogenic muscular atrophies (anterior horn celldegeneration, such as amyotrophic lateral sclerosis, infantile spinalmuscular atrophy and juvenile spinal muscular atrophy); Alzheimer'sdisease; Down's Syndrome; diffuse Lewy body disease; senile dementiarelated to Lewy body development; Wernicke-Korsakoff syndrome; chronicalcoholism; Creutzfeldt-Jakob disease; Subacute sclerosingpanencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica,and the like. Such a method can optionally comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one TNF antibody or specified portion or variant toa cell, tissue, organ, animal or patient in need of such modulation,treatment or therapy.

Other Therapeutic Uses of CD27-Neutralizing Antibodies

In addition to the above described conditions and diseases, the presentinvention also provides a method for modulating or treating fibroticconditions of various etiologies by modulating the immune-response toassociated or ancillary cells or cellular processes involving CD27 in,for example: liver fibrosis (including but not limited toalcohol-induced cirrhosis, viral-induced cirrhosis, autoimmune-inducedhepatitis); lung fibrosis (including but not limited to scleroderma,idiopathic pulmonary fibrosis); kidney fibrosis (including but notlimited to scleroderma, diabetic nephritis, glomerular pehpritis, lupusnephritis); dermal fibrosis (including but not limited to scleroderma,hypertrophic and keloid scarring, burns); myelofibrosis;neurofibromatosis; fibroma; intestinal fibrosis; and fibrotic adhesionsresulting from surgical procedures.

The present invention also provides a method for modulating or treatingor ameliorating the symptoms of an infectious disease in a cell, tissue,organ, animal or patient, by modulating the immune-response toassociated or ancillary cells or cellular processes involving CD27 in,for example: acute or chronic bacterial infection, acute and chronicparasitic or infectious processes, including bacterial, viral and fungalinfections, HIV infection/HIV neuropathy, meningitis, hepatitis (A, B orC, or the like), septic arthritis, peritonitis, pneumonia, epiglottitis,E. coli, hemolytic uremic syndrome, malaria, dengue hemorrhagic fever,leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis,gas gangrene, mycobacterium tuberculosis, mycobacterium aviumintracellulare, pneumocystis carinii pneumonia, pelvic inflammatorydisease, orchitis or epidydimitis, legionella, lyme disease, influenzaa, Epstein-Barr virus, vital-associated hemaphagocytic syndrome, vitalencephalitis/aseptic meningitis, and the like.

The contents of all cited references (including literature references,issued patents, published patent applications, and co-pending patentapplications) throughout this application are hereby expresslyincorporated by reference.

Other features of the invention will become apparent in the course ofthe following descriptions of exemplary embodiments which are given forillustration of the invention and are not intended to be limitingthereof.

4. Pharmaceutical Formulations

The invention provides for stable formulations of an CD27-neutralizingantibody, which is preferably an aqueous phosphate buffered saline ormixed salt solution, as well as preserved solutions and formulations aswell as multi-use preserved formulations suitable for pharmaceutical orveterinary use, comprising at least one CD27-neutralizing antibody in apharmaceutically acceptable formulation. Suitable vehicles and theirformulation, inclusive of other human proteins, e.g., human serumalbumin, are described, for example, in e.g. Remington: The Science andPractice of Pharmacy, 21st Edition, Troy, D. B. ed., Lipincott Williamsand Wilkins, Philadelphia, Pa. 2006, Part 5.

In order to form a pharmaceutically acceptable composition suitable foreffective administration, such compositions will contain an effectiveamount of the above-described compounds together with a suitable amountof carrier vehicle. Additional pharmaceutical methods may be employed tocontrol the duration of action. Controlled release preparations may beachieved through the use of polymers to complex or absorb the compounds.Another possible method to control the duration of action by controlledrelease preparations is to incorporate the compounds of the presentinvention into particles of a polymeric material such as polyesters,polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetatecopolymers. Alternatively, instead of incorporating these agents intopolymeric particles, it is possible to entrap these materials inmicrocapsules prepared, for example, interfacial polymerization, forexample, hydroxymethylcellulose or gelatin-microcapsules andpoly(methylmethacylate)-microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington supra (2006).

5. Administration of a CD27-Neutralizing Antibody

At least one CD27-neutralizing antibody in either the stable orpreserved formulations or solutions described herein, can beadministered to a patient in accordance with the present invention via avariety of delivery methods including intravenous (I.V.), intramusclular(I.M.); subcutaneously (S.C.); transdermal, pulmonary, transmucosal,using an formulation in an implant, osmotic pump, cartridge, micropump,or other means appreciated by the skilled artisan, as well-known in theart.

In one method of administering a CD27-neutralizing antibody, the drugsubstance is given intravenously from a previously installed catheterequipped with an infusion bag. The CD27-neutralizing antibody issupplied in 20-ml single-use vials, such as those supplied by ImmunoGen,Inc. (Cambridge, Mass.). Each vial contains protein at a concentrationof from 0.05 to about 2.0 mg/ml in a buffered solution (pH 6.5±0.5)comprised essentially of monobasic potassium phosphate (0.57 mg/ml),monobasic sodium phosphate monohydrate (0.20 mg/ml), dibasic sodiumphosphate (0.555 mg/ml), and sodium chloride (8.16 mg/ml) in purifiedwater, USP. The drug product is prefiltered twice upon instilling thedose volume into the infusion bag by passing it through a lowprotein-binding 5-μ filter and is administered to patients through aninline 0.22 μm filter within 8 h of preparation. After infusion, thei.v. line should be flushed with fluid to ensure delivery of the fulldrug dose.

In general, if administering a systemic dose of the antibody, it isdesirable to provide the recipient with a dosage of antibody which is inthe range of from about 1 ng/kg-100 ng/kg, 100 ng/kg-500 ng/kg, 500ng/kg-1 ug/kg, 1 ug/kg-100 ug/kg, 100 ug/kg-500 ug/kg, 500 ug/kg-1mg/kg, 1 mg/kg-50 mg/kg, 50 mg/kg-100 mg/kg, 100 mg/kg-500 mg/kg (bodyweight of recipient), although a lower or higher dosage may beadministered. Dosages as low as about 1.0 mg/kg may be expected to showsome efficacy. Preferably, about 5 mg/kg is an acceptable dosage,although dosage levels up to about 50 mg/kg are also preferredespecially for therapeutic use. Alternatively, administration of aspecific amount of the antibody may be given which is not based upon theweight of the patient such as an amount in the range of 1 ug-100 ug, 1mg-100 mg, or 1 gm-100 gm. For example, site specific administration maybe to body compartment or cavity such as intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,intralesional, vaginal, rectal, buccal, sublingual, intranasal, ortransdermal means.

The treatment may be given in a single dose schedule, or preferably amultiple dose schedule in which a primary course of treatment may bewith 1-10 separate doses, followed by other doses given at subsequenttime intervals required to maintain and or reinforce the response, forexample, at 1-4 months for a second dose, and if needed, a subsequentdose(s) after several months. Examples of suitable treatment schedulesinclude: (i) 0, 1 month and 6 months, (ii) 0, 7 days and 1 month, (iii)0 and 1 month, (iv) 0 and 6 months, or other schedules sufficient toelicit the desired responses expected to reduce disease symptoms, orreduce severity of disease.

6. Articles of Manufacture Comprising a CD27-Neutralizing Antibody

The invention includes an article of manufacture containing materialsuseful for the treatment of the disorders described above comprising aCD27-neutralizing antibody, a container and a label or package insert onor associated with the container. The article of manufacture preferablycontains at least one vial comprising a solution of at least oneCD27-neutralizing antibody with the prescribed buffers and/orpreservatives, optionally in an aqueous diluent, wherein said packagingmaterial comprises a label that indicates that such solution can be heldover a period of time. The invention may comprise an article ofmanufacture, comprising packaging material, a first vial comprisinglyophilized CD27-neutralizing antibody, and a second vial comprising anaqueous diluent of prescribed buffer or preservative, wherein saidpackaging material comprises a label that instructs a practitioner orpatient how to reconstitute the CD27-neutralizing antibody in theaqueous diluent to form a solution.

Suitable containers include, for example, bottles, vials, syringes, etc.The containers may be formed from a variety of materials such as glassor plastic. The container may have a sterile access port (for examplethe container may be an intravenous solution bag or a vial having astopper, optionally, capable of being pierced by a hypodermic injectionneedle).

At least one active agent in the composition is a CD27-neutralizingantibody. The label or package insert indicates that the composition isused for treating the indication of choice, such as SLE. The packageinsert herein may indicate that the antibody or composition is used totreat a condition that does not respond, or respond poorly, to treatmentwith the standard of care as outlined herein for specific diseases anddiagnoses. In other embodiments, the package insert may indicate thatthe antibody, antibody-conjugate or composition can be used also totreat a disease characterized by the need to modulate theimmune-response of cellular processes involving CD27.

Yet another aspect of the present invention is a kit for detecting CD27in a biological sample. The kit includes a container holding one or moreantibodies which binds an epitope of CD27 and instructions for using theantibody for the purpose of binding to CD27 to form an immunologicalcomplex and detecting the formation of the immunological complex suchthat the presence or absence of the immunological complex correlateswith presence or absence of CD27 in the sample. Examples of containersinclude multiwell plates which allow simultaneous detection of CD27 inmultiple samples.

While having described the invention in general terms, the embodimentsof the invention will be further disclosed in the following examples.

Example 1: CD27 Reagents and Methods

In order to generate and test CD27-binding monoclonal antibodies,protein constructs were generated which represent the full length ofhuman CD70 and human CD27 and the extracellular domain (ECD) of humanCD27.

Human CD27 (SEQ ID NO: 149) is a type 1 transmembrane protein comprisedof a signal peptide (from residues 1 to 20), extracellular (ECD, fromresidues 21 to 191), transmembrane (TM, from residues 191 to 212) andintracellular (ICD, from residues 213 to 260) domains. Human CD70 (SEQID NO: 2) is a type 2 transmembrane polypeptide of 193 amino acids inlength comprised of, from the N-terminus, an intracellular domain (ICD,from residues 1 to 17), transmembrane (TM, from residues 18 to 38) andextracellular domain (ECD, from residues 39 to 193). The complete CD70coding sequence was clonally expressed on the surface of HEK 293 cells.

For mAb ELISA and Proteon-based direct binding assays, amino acids 1-121of the CD27 ECD were transiently expressed in HEK293 cells with aC-terminal His6-tag peptide and purified by metal ion chromatography.For phage panning and ELISA assays, amino acids 1-173 of the ECD with aC-terminal His6-tag were HEK expressed and purified by metal ionchromatography followed by size exclusion chromatography on Superdex 75.Both of these CD27 proteins were biotinylated using NHS-ester chemistrytargeting amine residues on the protein. For crystallization, aminoacids 1-101 with a C-terminal His6-tag were expressed in a baculovirussystem and purified by metal ion chromatography by Proteose, Inc. Formouse immunization, CD27-Fc protein was purchased from R&D systems. Forsome studies, the complete CD27 coding sequence was clonally expressedon the surface of HEK 293 cells.

Human CD27 and human CD70 cDNA clones were ordered from Open Biosystems.Standard molecular biology techniques were used to generate expressionconstructs. Briefly, the open reading frames of the CD27 and CD70 geneswere PCR amplified and cloned into the mammalian expression vectors viarestriction endonuclease digestion and ligation, or via ligaseindependent cloning (LIC). Full length CD27 and CD70 genes were clonedinto the expression vector and were clonally expressed on the surface ofmammalian cells. The extracellular domain of CD27 was cloned intomammalian expression vectors and transiently expressed in HEK293 cellswith a hexa-his tail.

Example 2: GENERATION of CD27-NEUTRALIZING ANTIBODIES

Murine anti-human CD27 antibodies were generated by the hybridoma methodof Kohler and Milstein (1975). Ten 12-14 week old C3H/HeJ mice wereobtained from Charles River Laboratories. The mice were immunizedsubcutaneously (SQ) at the base of tail (BOT) with 50 microgm Hu CD27 Fc(R&D Systems) in combination with 0.33×10⁵ units each of murineinterferon-alpha and -beta (Biosource) in a final volume of 100 microLon day 1. On days 2 and 3, the mice were injected SQ BOT with theinterferons (same doses as on day 1). The mice were boosted with 50microgm Hu CD27-Fc in combination with 50 microgm anti-murine CD40agonist Mab (R&D Systems, MAB440) given SQ BOT in PBS on day 14; fourdays prior to splenic harvest for fusion.

For titer assessment, a capture phase EIA was performed. Briefly, plates(Nunc-Maxisorp) were coated with 0.1 microgram goat anti-ms Fc (JacksonImmunotech) in bicarbonate buffer overnight at 4° C. After blocking andwashing steps, dilutions of sera were added and plates were incubatedfor 30 minutes at RT. Following washing steps, the plates were incubatedfor 30 minutes at RT with 0.25 microgm/mL of biotinylated Hu CD27-ECD inblocking buffer and probed with HRP labeled Streptavidin (JacksonImmunotech) diluted 1:40,000 in 0.4% BSA/PBS of for 30 minutes at RT.Plates were washed as described above; then OPD (Sigma fast tabs)substrate solution was added, incubation for 10 minutes at roomtemperature, the color substrate development stopped by the addition of4N sulfuric acid at 25 microL/well, and the absorbance measured at 490nm.

A cell bank of the non-secreting BALB/c mouse myeloma fusion partner, FOwas purchased from ATCC (# CRL-1646). One frozen vial of FO cells wasthawed and resuspended in DMEM with Glutamax™ (modified) medium(Invitrogen) supplemented with 10% (v/v) FBS (Hyclone). The cells wereexpanded, cryopreserved and deemed sterile and free of mycoplasma byCharles River Laboratories. The C1833A (Centocor) cell line was alsoused in this fusion. This cell line was derived in-house by knockingdown expression of the CHOP gene in the FO cell line so it requiresgrowth under selection with geneticin. Cells were treated as FO's abovewith the exception of growing in DMEM with Glutamax™ (modified) mediumsupplemented with 10% (v/v) FBS (Hyclone) and 500 ug/mL of geneticin(Gibco). Both the FO and C1833A cell lines were subjected to cellsynchronization prior to fusion. Briefly, 1.5-2×108 cells were seededinto 180 mL of DMEM with Glutamax™ (modified) medium supplemented with0.25% (v/v) FBS (Hyclone) and incubated at 37° C. for 13 hours. Anadditional 20 mL of FBS was added for a final FBS concentration of 10%and incubated for an additional 13 hours at 37° C. prior to use. C1833Acells were constantly under geneticin selection throughout cellsynchronization process. The myeloma cells were washed in PBS, counted,and viability determined (>78%) via Guava Viacount software prior tofusion.

On the day of fusion, the animals were euthanized by CO₂ asphyxiation.The spleens were removed aseptically and immersed in 10 mL of coldphosphate-buffered saline (PBS) containing antibiotics (PSA) (Sigma).

A single cell suspension of splenocytes was prepared and subjected toRBC lysis using RBC lysis buffer (Sigma). Washed cells were labeled formagnetic sorting as per the manufacturer's instructions, usinganti-murine Thy1.2, anti-murine/human CD11b and anti-murine IgM magneticbeads (Miltenyi Biotec #130-049-101, 130-149-601 and 130-047-301respectively) and then sorted using the AutoMacs Pro instrument byrunning the Deplete program. Both the unlabeled (plasmablast B cellenriched) and labeled cell fractions were collected then counted via theGuava PCA. Positively labeled cells were discarded. Unlabeled cells weredivided in half for fusion to both FO and C1833A fusion partners.Fusions were carried out at a 1:1 ratio of murine myeloma cells toviable spleen cells according to the method of De St. Groth (JImmunological Methods. 35:1-21. 1980). Briefly, spleen and myeloma cellswere mixed together, pelleted and washed once in 50 mL of PBS. Thepellet was resuspended with 1 mL of polyethylene glycol (PEG) solution(2 g PEG molecular weight 4000, 2 mL DMEM, and 0.4 mL DMSO) at 37° C.over 30 seconds. The cell/fusion mixture was then immersed in a 37° C.water bath for approximately 60 seconds with gentle agitation. Thefusion reaction was stopped by slowly adding 37° C. DMEM over 1 minute.The fused cells were allowed to rest or 5 minutes at room temperatureand then centrifuged at 150×g for 5 minutes. Cells were then resuspendedin HAT medium [DMEM with Glutamax™ (modified), supplemented with 20%FBS, 5% Origen, 25 microg/mL gentamicin (Sigma) and HAT (100 microMhypoxanthine, 0.4 microM aminopterin, and 16 microM thymidine (Sigma),and seeded in 96-well flat bottom polystyrene tissue culture plates(Corning #3997) or methylcellulose medium (StemCell Technologies,MediumD cat#03804) containing ˜2.25 μg/mL of AF488 human CD27 (JanssenResearch & Development, LLC). Plates were incubated in a humidified 37°C. incubator with 7% CO₂ for 7-10 days. Single colonies were selectedfrom methylcellulose plates for screening utilizing the ClonepixFL orunder a white light microscope.

Example 3: Bioactivity of Recombinant Mabs

The ability of the binding domains from the murine antibodies to bindCD27 and to block certain bioactivities of CD27 was analyzed usingvarious in vitro assays as described below.

A solid phase EIA was used to screen the hybridoma supernatants forantibodies capable of binding human CD27. Plates (Nunc-Maxisorp #446612)were coated overnight with 4 μg/mL Fab goat anti-huFc (Jackson#109-006-098) in Bicarbonate buffer O/N at 4° C. Without washing, thewells were blocked with 200 microL of 0.4% (w/v) bovine serum albumin(BSA) in PBS for 1 hr at RT. After washing with 0.15 M saline containing0.02% (w/v) Tween 20, 50 microl of huCD27-Fc in 0.4% BSA/PBS was addedto the plates for 1 hr at RT. After washing again, 50 microl ofundiluted hybridoma supernatants were incubated on coated plates for 30minutes at RT. Plates were washed three times and then incubated with 50microL of goat anti-murine Fc HRP (Jackson #115-036-071) diluted1:10,000 for 30 minutes at RT. Plates were again washed and developed asdescribed above for titer assessment. For assessment relative bindingcapacity of hybridoma Mabs similar assay was performed using Maxisorp384 well plates (NUNC 464718) with serially diluted hybridomasupernatants (normalized to a starting concentration of 5 microg/mL.This assay identified 386 positive hybridomas.

All 386 CD27 specific hybridomas were screened for the ability toinhibit binding of huCD27 to huCD70 using biochemical binding assayswith IM-9 cells, a B-lymphoblastoid cell line found to endogenouslyexpressing human CD70. Maxisorp plates (VWR #62409-314) were coated withrecombinant human CD27/Fc (R&D Systems, Cat#382-CD) at 250 nanogram(ng)/mL and incubated overnight at 4° C. The next day plates wereblocked with blocking buffer (Pierce, Cat#37543) and then washed withwash buffer I, that contains PBS without Ca++ or Mg++, 0.01% Tween-20.Controls (mouse MAB to hCD27, R&D Systems, Cat# MAB382; Mouse IgG1isotype control, R&D Systems, Cat# MAB002; mouse IgG2a isotype control,R&D Systems, Cat# MAB003) were included on each plate. 50 uL/well ofhybridoma samples or controls were mixed with 50 μL/well of harvestedIM-9 cells, human B-lymphoblastoid cell line (ATCC, CCL-159) and wereincubated for 1 hour at RT without shaking. At the end of incubation,plates were washed with wash buffer II, to remove all unbound cells, andthen lysed with 50 uL/well of Cell Titer Glo reagent (Promega, Cat#G7571). After 10 minutes incubation with shaking, plates were read onEnvision (PerkinElmer, 2102 Multilabel reader). The luminescent signalgenerated is proportional to the amount of ATP present and directlycorrelated to the number of live cells present in the well captured byCD27 binding. Based on the results of the biochemical binding assay,about 50% of CD27 specific clones were neutralizing.

To eliminate redundancy among the neutralizing clones, competitionbinding assays were performed to bin the antibodies into competitiongroups. In this assay, hybridoma supernatants were assessed individuallyas both capture and detection reagents with each of the positivehybridomas in the panel. Antibodies forming effective capture/detectionreagents with each other likely recognize spatially-separated epitopeson the CD27 protein, thus allowing both antibodies to bind to the targetprotein at the same time. Groups of clones exhibiting similar patternsof activity across the entire panel likely bind to similar epitopes.Selecting clones from different groups therefore provided antibodiesrecognizing different epitopes. Briefly, 384 well Nunc Maxisorp plates(464718) were coated with goat anti-mouse Fc (JIR115-005-071) in coatingbuffer overnight at 4° C. Plates were then blocked with 0.4% BSA in PBSfor 30 minutes at room temperature. At this step and all subsequentsteps plates are washed with PBS, 0.02% Tween-20. Each well of a row(one row per supernatant) received 20 uL of supernatant (neatsupernatant was used for the initial screen but for rescreening thesubclones, supes were normalized to 2 ug/ml of mAb) was along withcontrols (mouse anti-huCD27, R&D Systems, Cat# MAB382; mouse isotypecontrol Cat#555439, Becton-Dickenson) then incubated for 30 minutes atRT. After washing, 25 uL of unlabeled Hu CD27-ECD-His-tag was preparedin PBS plus 10% mouse sera (Bioreclamation mouse serum CD-1lot#MSEBREC.18565) at 0.3 (or 0.8 for concentration normalized)microg/ml was added to all wells, followed by 30 minutes incubation atroom temperature then washed. Each supernatant was added down a singlecolumn and incubated for 30 minutes at RT with 25 uL of a mixtureprepared as follows: (—pre-incubate supernatants with goat anti-mouse FcHRP (Jackson 115-036-008), by mixing 150 uL of 1:1000 goat anti-mouse FcHRP with each 1000 uL of supernatant (for the primary screen) or 90 uLof 1:2000 per 600 uL of supernatant adjusted to 2 ug/mL: (forrescreening subclones). After 30 minutes incubation at room temperature,add 200 microL of 100% normal mouse sera per mL and incubate anadditional 30 minutes at RT. Plates were washed then incubated for 15minutes at RT with 100 uL/well of citrate-phosphate substrate solution(0.1 M citric acid and 0.2 M sodium phosphate, 0.01% H2O2, and 1 mg/mLOPD). Substrate development was stopped by addition of 25 uL of 4Nsulfuric acid and the absorbance measured at 490 nm using an automatedplate reader. This binning assay identified three groups that recognizenon-overlapping binding sites on huCD27 antigen. Selected antibodiesfrom all three groups were scaled up for antibody production,purification and further testing in functional assays.

Inhibition of Cell Signaling

Binding of CD70 to CD27 induces signaling that leads to downstreamactivation of the transcription factor, NF-kβ. A NF-kβ reporter assaywas established for further antibody characterization. The assay was runin two modes: (1) to assess antibody antagonism by neutralization ofCD70 induced CD27 activation and (2) to assess antibody agonism byactivation of CD27 signaling without CD70 ligation. HEK-293F cells weretransfected with a total of 36 ng of DNA containing both human CD27 andluciferase constructs, under control of the NF-kβ promoter. HEK-293Ftransfectants were plated 5×10⁴ cells per well in 40 uL Freestyle media(Gibco) in 96-well plates. Dilutions of CD27-neutralizing hybridoma mAbswere added to the assay plate in Freestyle media (Gibco) for a finalconcentration of 50 ug/mL with 1:3 dilutions and plates were incubatedat 37° C. (95% O₂/5% CO₂) for one hour. To test for ability of hybridomamAbs to neutralize CD70:CD27 signaling, terminally irradiated (4000rads) HEK-293E CD70 episomal cells were added at 20% of the number ofCD27 transfectant cells to the assay plate. To test for agonist activityof hybridoma mAbs, addition of CD70 episomal cells was omitted. Assayplates were incubated overnight at 37° C. (95% O₂/5% CO₂) and developedusing the Steady-Glo® Luciferase Assay System (Promega) according to theinstructions of the manufacturer. Four CD27-neutralizing hybridoma mAbs,C2177, C2186, C2191, and C2192, that dose-dependently blocked theCD70-mediated CD27 signaling without causing significant dose-dependentagonistic activation of the CD27 receptor in the absence of CD70stimulus were selected for further characterization. The IC₅₀s forblocking IM-9 cell binding to CD27 and CD70-mediated signaling in NF-kβreporter gene assay are summarized for these four antibodies in Table 1.Agonism activity in the NF-kβ reporter gene assay is shown as foldincrease in CD27 signaling relative to an irrelevant isotype controlantibody (mouse IgG1 to rat EMP protein) in the absence of CD70 stimulusat the maximum tested concentration of antibody.

Affinity for CD27

The Kms of antibodies C2177, C2186, C2191, and C2192 for monomericsoluble CD27 at 25° C. were measured by Biacore and are reported inTable 1. Assays were carried out on a BIACORE 3000 (BIAcore, Inc.)surface plasmon resonance (SPR) instrument. The samples were prepared inDulbecco's phosphate buffered saline pH 7.4 containing 0.005% surfactant(polysorbate 20). Goat anti-mouse Fc specific antibody (JacksonImmunoresearch laboratories Prod #115-005-071) was covalently attachedto carboxymethyl dextran coated gold surfaces (CM-5 Chip, Biacore).Prior to immobilization the chip was pretreated with 50 mM NaOH, 100 mMHCl and 0.1% sodium dodecyl sulfate with injection of deionized water inbetween the pre-treatments. The antibodies were diluted with 10 mMsodium acetate buffer pH 4.5 and coupled to the carboxymethylateddextran surface of the chip using the manufacturer instructions foramine-coupling chemistry. The remaining reactive groups on the surfacewere deactivated using ethanolamine-HCl. The mAb were captured on thesensor surface via Fc domain. The associations of human CD27 ECDinjected at increasing concentrations (0.6-150 nM, 4-fold dilutionseries) were monitored for three minutes and the dissociations for tenminutes. Regeneration of capture surfaces to baseline was optimizedusing two 3 second pulses of 100 mM phosphoric acid. Data were processedusing the Scrubber software, version 1.1 g (BioLogic Software). Doublereference subtraction of the data was performed to correct for buffercontribution to the signal and instrument noise. The kinetic analysis ofthe processed data was carried out using the Biaevaluation 4.0.1software (GE Healthcare Bio-Sciences, Uppsala, Sweden). Binding profileswere described by a 1:1 binding model indicating a monovalent binding ofCD27.

TABLE 1 IM-9 Biacore Binding¹ NF-kβ Reporter K_(D) IC₅₀ IC₅₀ ² Agonism³mAb Isotype nM ug/ml ug/ml at 50 ug/ml C2177 mIgG1 3.07 0.063 0.0401.850 C2186 mIgG1 2.55 0.059 0.105 2.547 C2191 mIgG1 2.62 0.059 0.0803.053 C2192 mIgG2a 0.21 0.054 0.232 5.394 ¹IC₅₀ for mAb inhibition ofIM-9 cells binding to immobilized CD27 ²IC₅₀ for mAb inhibition in thereporter gene assay ³Agonist activity of mAbs at 50 ug/ml in the absenceof CD70, measured as fold-increase in reporter gene signal relative toisotype control antibodies.Inhibition of Cell Proliferation

The proliferation of T-cells sub-optimally activated in culture withanti-CD3 plus anti-CD28 antibodies is enhanced by CD70 ligation of CD27expressed on the T-cells. The four murine neutralizing antibodies wereassessed for their ability to inhibit T-cell proliferation in thepresence of CD70 and to induce proliferation in the absence CD70. FrozenCD4+ T cells were purchased from AllCells, LLC. Cells were thawed andplaced into IMDM medium containing 10% FBS, 1% 1-glutamine, and 1%Penicillin-Streptomycin. Prior to plating cells, anti-CD3 (OKT3)antibody was coated onto a U-bottom plate at 1 ug/□mL in PBS overnightat 4° C. Cells were counted, brought to a concentration of 1×10⁶cells/mL, and plated at 1×10⁵ cells/well. Soluble anti-CD28 was added asa secondary activation signal at 1 ug/mL per well. Irradiated (6000rads) HEK cells transfected with either human CD70 or vector alone(mock) were added to appropriate wells at 2×10⁴ cells/well (20%). Cellswere stimulated for 3 days, 0.9 uCi thymidine [methyl-3H] was added toall sample wells and the cells were incubated for 18-24 hours. On thefourth day of stimulation, cells were harvested onto a filter plateusing the PE Filtermate Harvester. The plate was allowed to dry and 30uL of MicroScint™-20 was added to all sample wells. The plate was readon a PE TopCount NXT, and data collected was as CPM. Antibody C2177shows dose-dependent inhibition of CD70 mediated T-cell proliferationand very weak intrinsic agonistic activity in the absence of CD70 (FIG.1). Similar results were observed for the C2186, C2191 and C2192antibodies. The IC₅₀ and maximal % inhibition for these antibodies arereported in Table 2. None of the antibodies showed consistentstimulation of proliferation in the absence CD70 ligation, indicating alack of intrinsic agonist activity.

In addition, the C2177, C2186, C2191 and C2192 antibodies showeddose-dependent inhibition of CD70-mediated T-cell proliferation asmeasured in a CSFE assay with no effect on proliferation in the absenceof CD70 stimulus. Frozen CD3+ T cells were purchased from AllCells, LLC.Cells were thawed and placed into IMDM medium containing 10% FBS, 1%L-glutamine, and 1% Penicillin-Streptomycin. Cells were pre-labeled with2.5 mM CFSE (Invitrogen), quenched with FBS and washed with T cellmedia. CFSE is a dye that passively diffuses into cells and becomehighly fluorescent upon binding with intracellular amines. Upon celldivision each daughter cell will contain half of the CFSE label of theparental cell, thus cell proliferation may be monitored by trackingnumbers of cells with different CFSE intensity. The cells were broughtto a concentration of 1×10⁶ cells/mL, and plated at 1×10⁵ cells/well.Prior to plating cells, anti-CD3 (OKT3) antibody was coated onto aU-bottom plate at 0.5 ug/mL in PBS overnight at 4° C. Soluble anti-CD28was added as a secondary activation signal at 0.1 ug/mL per well.Irradiated (6000 rads) HEK cells transfected with either human CD70 orvector alone (mock) were added to appropriate wells at 2×10⁴ cells/well(20%). Cells were stimulated for 4 days and analyzed by FACS analysis tocount divided cells containing different intensity levels of CFSE label.

Inhibition of Plasma Blast Differentiation

The CD27-neutralizing hybridoma mAbs were also tested in a plasma blastdifferentiation assay with primary human B-cells. CD19+ human Blymphocytes that had been negatively selected from peripheral blood ofnormal donors (obtained from AllCells) were cultured for 6 days in thepresence of either 1 ug/mL anti-CD40 antibody (clone MAB89, Abcam) and100 ng/mL Interleukin 21 (Invitrogen) or 1 ug/mL soluble humanrecombinant CD40 ligand and 2 ug/mL ‘enhancer for ligands’ (both AlexisBiochemicals) and 100 ng/ml Interleukin 21 in 96-well plates at 10⁵ Bcell per well. CD27-neutralizing hybridoma or isotype control antibodieswere added in the presence or absence of 2×10⁴ irradiated (6000 rads)CD70-expressing HEK 293 cells or MOCK-transfected HEK 293 cells.CD27-neutralizing hybridoma mAbs and matching isotype controls were usedat 25, 2.5, and 0.25 ug/mL. On day 6, cell samples were analyzed by flow□cytometry and fractions of plasma blasts were identified as forwardscattering/high, IgDminus, CD38bright, CD20low. The effect ofCD27-neutralizing mab was calculated as plasma blast frequency in B-cellcultures containing CD70 expressing cells and hybridoma mabs normalizedto plasma blast frequency in corresponding B-cell cultures containingmock-transfected cells. The percent inhibition by the C2177, C2186,C2191 or C2192 mAbs at 2.5 ug/mL is shown in Table 2. Agonistic activityin the absence of CD70 stimulus was not observed for any of these mAbs.

TABLE 2 T-Cell Proliferation - CD4+ Cells Plasma blast % Inhibition athighest differentiation IC₅₀ concentration (30 ug/ml) % Inhibition at2.5 ug/ml MAb ug/ml (Mean ± SEM) (Mean ± SEM) C2177 0.245 75.4 ± 4.1 (n= 6, 2 donors)  78 ± 20 (n = 4) C2186 0.775 65.3 ± 4.4 (n = 6, 2 donors) 93 ± 27 (n = 4) C2191 0.3 58.3 ± 6.0 (n = 6, 2 donors) 105 ± 9 (n = 4)C2192 0.445 75.7 ± 5.7 (n = 6, 2 donors)  74 ± 0 (n = 2)

Example 4: Epitope Mapping and Grouping

To more carefully evaluate the initial binning, competition assays werecarried out with some of the purified neutralizing mAbs and with theCD27-neutralizing antibody, MAB 382 (R&D Systems). Briefly, 5 μl (10μg/mL) of CD27-Fc chimeric protein (R&D Sysytems, Cat#382-CD) was coatedon a MSD HighBind plate (Meso Scale Discovery, Gaithersburg, Md.) perwell for 2 hr at RT. 5% MSD Blocker A buffer (Meso Scale Discovery,Gaithersburg, Md.) was added to each well and incubated for 2 hr at RT.Plates were washed three times with 0.1 M HEPES buffer, pH 7.4, followedby the addition of a mixture of 10 nM labeled CD27 antibody withdifferent concentrations of a competitor antibody (1 nM to 2 uM).Antibodies were labeled with MSD Sulfo-Tag™ NHS-ester, an amine-reactiveN-hydroxysuccinimide ester which couples to primary amine groups ofproteins to form a stable amide bond. After a 2-hour incubation withgentle shaking at RT, plates were washed 3 times with 0.1M HEPES buffer(pH 7.4). MSD Read Buffer T was diluted with distilled water (4-fold)and dispensed at a volume of 150 μL/well. The plates were analyzed usinga SECTOR Imager 6000 which detects electrochemiluminescence throughSulfo-Tag labels that emit light upon electrochemical stimulationinitiated at the electrode surfaces of MSD microplates.

The competition studies defined three competition groups for theantibodies summarized in Table 3, confirming the initial binning assays.C2179, C2192 and MAB382 constitute one group; C2177, C2182, C2186 andC2193 are a second group; and C2191 constitutes a separate group.

TABLE 3 Labeled Antibody Competitor C2179 C2177 C2182 C2186 C2191 C2177− + + + − C2179 + − − − − C2182 − +/− + + − C2186 − + + + − C2191 − − −− + C2192 + − + + − C2193 − +/− + + − MAB382 + − − − −

Example 5: Epitope and Paratope Identification by X-Ray Crystallography

The detailed epitopes and paratopes of antibodies C2177 and C2191 weredetermined by co-crystallization of their corresponding Fabs with CD27ECD fragment (residues 1-101) as a trimeric complex and structuredetermination by X-ray crystallography. The His-tagged chimeric versions(mouse variable domain, human constant domain) of C2177 Fab and C2191Fab were expressed in HEK293 cells and purified using affinity and sizeexclusion chromatography. The His-tagged ECD fragment (residues 1-101)of human CD27 was further purified by anion exchange chromatography. Theternary complex CD27:C2177 Fab:C2191 Fab was prepared by mixing CD27with the excess of Fabs at a molar ratio 1:1.25:1.25. The complex wasincubated for 2 h at 4° C., separated from the uncomplexed species usingsize-exclusion chromatography, and concentrated to 12 mg/mL in 20 mMTris pH 8.5, 250 mM NaCl. Crystallization of the complex was carried outby the vapor-diffusion method in sitting drops at 20° C. The crystals ofthe complex were obtained from 24% PEG 3350, 0.2 M ammonium chloride,0.1 M Tris buffer, pH 8.5. For X-ray data collection, one crystal wassoaked for a few seconds in a cryo-protectant solution containingcrystallization solution supplemented with 20% glycerol, and flashfrozen in the stream of nitrogen at 100 K. Diffraction data werecollected at the Rigaku MicroMax™-007HF X-ray generator equipped with aSaturn 944 CCD detector and an X-stream 2000 cryocooling system (Rigaku)over a 240° crystal rotation with 2-min exposures per 0.25°-image andwere processed with the program XDS (Kabsch W. 2010. Acta Crystallogr.D66:125-132). The crystals belong to the monoclinic space group P21 withunit cell parameters: a=141.1 Å, b=53.0 Å, c=143.4 Å, α=90°, β=112.2°,γ=90°.

The crystal structure of the ternary complex was determined at 3.5 Åresolution and refined to the crystallographic R-factor of 26%. The Fabsof C2177 and C2191 bind CD27 at spatially distinct non-overlappingepitopes (FIG. 2). C2177 Fab binds the N-terminal (distal from the cellsurface) portion of CD27. The epitope covers 700 Å2 and includes 9residues: K5, S6, P8, H11, W13, G16, K17, H36, R37 (FIG. 3). Theparatope is defined as antibody residues in contact (within 4 Å) withthe antigen. The C2177 paratope includes 5 residues from VL (Y31, Y36,Y53, N57, N96) and 9 residues from VH (S31, W33, Y52, D55, D57, Y101,Y102, D104, Y105) (FIG. 3). All 6 CDRs are involved in antigenrecognition. H36 and R37 are the central residues of the epitope. Theystack against Y31 of VL and Y102 of VH; H36 also forms a salt bridge toD104 of VH.

C2191 Fab binds CD27 at the ‘side’ surface (FIG. 2) and covers 800 Å2 ofthe surface. The epitope includes 10 residues: F28, D43, P44, 146, P47,G48, V49, H60, S63, H66. The C2191 paratope includes 7 residues from VL(Y34, F36, Y53, L54, R96, L98, W100) and 8 residues from VH (S31, Y32,Y50, N57, Y59, R100, G101, N102) (FIG. 4). The antibody-antigeninteractions are dominated by the hydrophobic interactions betweenresidues 44-49 of CD27 and a hydrophobic patch at the VL CDRs.

The different location of the C2177 and C2191 epitopes suggestsdifferent mechanisms of action of these antibodies. C2191 probablydirectly competes with CD70 ECD for the overlapping epitopes on the‘side’ surface of CD27. C2177 antibody, on the contrary, does notcompete for the same epitope but rather prevents the approach of thecells bearing CD27 and CD70. This observation is supported by the factthat C2191 prevents binding of soluble CD70 ECD to CD27 whereas C2177does not.

Example 6: Antibody Modulation of Human Lymphocyte Response

An immune deficient mouse model, NOD/SCID-IL2Rγ^(null) (NSG) mice, wasdeveloped to study aspects of the human immune system control by T-cellresponses (Markus G Manz & James P Di Santo Renaissance for mouse modelsof human hematopoiesis and immunobiology Nature Immunology 10, 1039-1042(2009)). Adoptive transfer of human PBMCs into immune-compromised (NSG)mice was employed to evaluate the effects of an anti-CD27 antibody onhuman cell engraftment and/or proliferation. The model allows theevaluation of the effects of targeting human CD27 on antibody productionand T-cell mediated responses.

Antibodies C2177 and C2191 were administered at the time of celltransfer and then twice a week for 3 weeks. On day 21, the mice weresacrificed, cells were purified from blood and spleen and subsequentlycharacterized by flow cytometry. CTLA4-Ig (Orencia, BMS) was included asa positive control for immune suppression. Human cellengraftment/expansion was measured by evaluating the presence of humanCD45⁺ cells in the blood and spleen samples.

The mice were closely monitored and the time of sacrifice was determinedbased on XGVH symptoms in accordance with animal welfare guidelines. Theexperimental readouts used to evaluate the effects of anti-CD27treatment included: body weight (twice weekly), observable signs of XGVH(twice weekly), such as posture, activity level, grooming, skin lesions(in particular, around the eyes and ears) using 1-5 score system,absolute count of human cell subsets and activation status using flowcytometric analysis of (1) human PBMC injected, (2) mouse PB(once/week), and (3) spleen and bone marrow; determination of totalhuman Ig, IgM and IgG in serum, spleen and BM using an ELISA, and, uponsacrifice, histology or immunohistochemistry to determine the levels ofhuman infiltration in target organs, such as liver, kidney, lung andspleen.

The treatment groups were as follows:

1. PBMC (20 to 40 million cells per mouse, i.p.)

2. PBMC+CTLA4-Ig (10 mg/kg)

3. PBMC+Isotype control antibody, 2×/week for 3 weeks

4. PBMC+anti-CD27 antibody 2×/week for 3 weeks

Mice dosed with 10 mg/kg anti-CD27 mAbs, C2177 and C2191, (hybridomaantibodies chimerized on a human IgG4 (ala/ala, ser->pro) scaffold) hadstatistically significant fewer human CD45⁺ cells when compared to PMBCalone or isotype control in PMBCs isolated from the blood or spleensamples.

Example 7: Human Framework Adaptation of the C2177 and C2191 MABS

The antigen-binding site and the regions used to transfer the antigenspecificity from antibodies C2177 and C2191 into the human FR's werereclassified as outlined in Raghunathan G. US20090118127 A1, 2009. Inbrief, the antigen-binding regions have been defined using various terms(review in Almagro and Fransson, Front Biosci 13: 1619-1633, 2008). Theterm “Complementarity Determining Regions (CDRs)” is based on sequencevariability (Wu and Kabat, J. Exp. Med. 132:211-250, 1970). There aresix CDRs; three for V_(H) (H-CDR1, H-CDR2, H-CDR3), and three for VL(L-CDR1, L-CDR2, L-CDR3) (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991). “Hypervariable regions,”“HVR's,” or “HVL's” refers to the regions of an antibody variable domainwhich are variable in structure as defined by Chothia and Lesk (Chothiaand Lesk, Mol. Biol. 196:901-917, 1987). There are six HVR's, three forVH (H1, H2, and H3) and three for VL (L1, L2, and L3).

In the HFA method, the regions targeted for transferring the specificityof the non-human antibody into the human FRs (HFRs) are the CDRs asdefined by Kabat (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md., 1991) except in the region corresponding to the CDR-1 ofV_(H). For this region a combination of CDR and HVL (extended CDR-1 ofV_(H)) are transferred from the non-human antibody into the human FRs(as provided in Tables 30, 31, 34, 35). In addition, variants with ashorter transferred CDR-H2 (called Kabat-7 [Raghunathan G. US20090118127A1, 2009]) are generated and tested.

Human FR Selection.

Human FRs, defined as the regions in the V regions not comprised in theantigen-binding site, were selected from the repertoire of functionalhuman germilne IGHV, IGKV, IGKJ and IGHJ genes. The repertoire of humangermline gene sequences was obtained by searching IMGT database (Kaas,et al., Nucl. Acids. Res. 32, D208-D210, 2004; Lefranc M.-P et al.,Nucl. Acids Res., 33, D593-D597, 2005) and compiling all “01” alleles asof Oct. 1, 2007. From this compilation, redundant genes (100% identicalat amino acid level) and those with unpaired cysteine residues wereremoved from the compilation.

Initial selection of human sequences for HFR was based on sequencesimilarity of the human IGHV germline genes to the entire length of themouse V_(H) region including FR-1 to 3 as well as H-CDR-1 and H-CDR-2.In the next stage, the selected human sequences were rank ordered usinga score that takes into account both the length of the CDRs and sequencesimilarities between CDRs of mouse and human sequences. A standardmutation matrix, such as the BLOSUM 62 substitution matrix (Henikoff andHenikoff, Proc Natl Acad Sci USA. 89, 10915-9, 1992) was used forscoring alignments of the CDRs of mouse and human sequences and a largepenalty was applied if there was an insertion and/or deletion in the CDRloops. FR-4 was selected based on sequence similarity of the IGHJgermline genes (Kaas, et al., Nucl. Acids. Res. 32, D208-D210, 2004;Lefranc M.-P et al., Nucl. Acids Res., 33, D593-D597, 2005) with mouseantibodies C2177 and C2191 sequences. A similar procedure was used tochoose human FRs for V_(L). IGVK, germline genes were used for selectingFRs 1-3 and L-CDR 1-3. IGJK germline genes were used for selecting FR-4.

In addition to sequence criteria, a 3D homology model for the Fvfragments was constructed using Modeler (Sali and Blundell. J. Mol.Biol. 234: 779, 1993) in the program suite from Accelrys, Inc. Themodels were utilized for analysis of the HFR variants, including CDRcharacterization and assessment of developability liabilities.Additional considerations for selection of HFR variants were to minimizethe number of exposed methionine and tryptophan residues, eliminatepotential N-glycosylation sites and to favor human germlines with thehighest expression profile in silico (de Wildt, J. Mol. Biol. 185: 895,1999).

For path 1 framework adaptation and optimization of C2177, six V_(H) andfour V_(L) HFR variants were included in the library. The V_(H) andV_(L) HFR variants were paired in a combinatorial manner to yield 24 HFRvariant pairs plus 10 controls pairing all HFR variants with thecounterpart V region of C2177 plus the parent C2177 itself to give atotal of 35 combinations. Similarly for C2191, five V_(H) and four V_(L)HFR variants were paired in a combinatorial manner to yield 20 HFR plus9 controls combining all HFR V variants paired with the counterpart Vregion of C2191 plus the parent C2191 parent itself for a total of 30variants. DNA encoding the selected variable domains was recombinedusing standard methods to assemble complete MAbs with human IgG1 andkappa constant regions. The resulting reference chimeric antibody ofC2177, designated M40, is comprised of variable regions H7 and L18. Thecorresponding chimeric antibody of C2191, designated M41, is comprisedof variable regions H10 and L20. The mAbs were transiently expressed in48-well plates in HEK 293E cells. Supernatant fluid from the cultureswas tested for expression and binding activity 96 hours followingtransfection. The expression level of secreted mAb was evaluated usingOctet technology to measure the rate of antibody binding to Protein Abiosensors. The expression level was quantified by comparison tostandard samples of known antibody concentration. An 8-point standardcurve consisting of a 1:2 serial dilution of antibody of the identicalisotype, was assembled, starting at 100 ug/ml. Biosensors were hydratedfor 10 minutes in spent medium, and the binding rate of standards andunknown samples was measured for 2 minutes. Data was analyzed using the5 parameter weighted dose-response equation and the initial slopebinding rate algorithm. Samples with expression >1 ug/ml were diluted to1 ug/ml with spent medium and screened using a single point ELISA. Forthis ELISA, 96 well black maxisorp plates were coated with 50 uL of 3ug/ml goat anti human IgG FC diluted in carbonate-bicarbonate buffer, pH9.4 at 4 C overnight and then washed three times with wash buffer (PBSwith 0.05% Tween-20), blocked with 300 μl StartingBlock (ThermoScientific) solution for 1 hour, then washed as before. Samples orstandards were diluted to 100 ng/ml in spent medium, and 50 ul was addedto the assay plate at room temperature for 1 hour with shaking. Theplates were washed thrice and 50 ul per well of human CD27 ECD with HisTag was added at 60 ng/ml diluted in Assay Buffer (PBS with 1% FBS and0.05% Tween-20) and incubated for 1 hour at room temperature. Afterwashing, 50 ul per well of Qiagen peroxidase conjugated penta-his at1:2000 dilution in assay buffer was added and incubated 1 hour at roomtemperature with shaking. The BM ChemiLum Substrate (BM Chemilum, POD,Roche) was mixed per manufacturer's instructions, and 50 ul was added tothe plates after a final wash. After 10 minutes the plates are read onPerkin Elmer Envision Reader.

The results of screening the C2177 combinatorial library showed that allV-regions bind to CD27 with varying strengths. Several HFR variants gavea higher binding signal than the parent C2177 while others showedbinding that was comparable or lower than the parent. All VLs boundantigen at detectable levels and did not influence binding HFR variantsexpressed at acceptable but lower levels than parent. Twenty-four of theC2177 HFR antibodies (VH, VL combinations) showed CD27 binding andexpression >1 ug/ml.

The results of screening the C2191 combinatorial library showed that allexcept one VH bound to CD27 with varying signals. With the exception ofpairing with this VH, all VLs showed binding to CD27. Several HFRvariants gave a higher binding signal than the parent C2177, whileothers showed binding that was comparable or lower than the parent.Seventeen C2191 HFR antibodies (VH, VL combinations) demonstrated CD27binding and expression >1 ug/ml.

Based on relative binding affinity for CD27 measured by ELISA, fifteenC2177 and eleven C2191 variants were chosen for pilot-scale expressionand purification. Pilot-scale expression was done transiently in CHO-Scells at a volume of 750 ml. The harvested supernatants were purifiedvia Protein A chromatography and the purified proteins were assessed fortheir affinity and functional activity.

The affinities of the HFR C2177 human MAb variants were measured bySurface Plasmon Resonance (SPR) using a ProteOn XPR36 proteininteraction array system (BioRad). The rates of CD27 association anddissociation were measured for each variant. The biosensor surface wasprepared by covalently coupling Goat anti-Human IgG (Fc) antibodies tothe surface of a GLC chip (BioRad) using the manufacturer instructionsfor amine-coupling chemistry. Approximately 5,000 RU (response units) ofantibody were immobilized. The kinetic experiments were performed at 25°C. in running buffer (PBS, 0.01% P20, 0.01% BSA). 1:3 serial dilutionsof human CD27 ECD from, starting at 300 nM were prepared in runningbuffer. About 350 RU of mAb were captured on each channel of the sensorchip. An isotype-matched antibody control was immobilized in channel 6and used as a reference surface. Capture of mAb was followed by threeminutes injection (association phase) of antigen at 30 uL/min, followedby 10 minutes of buffer flow (dissociation phase). The chip surface wasregenerated by injection of 0.85% phosphoric acid at 100 uL/min. Datawas processed on the instrument software. Double reference subtractionof the data was performed by subtracting the curves generated by bufferinjection from the reference-subtracted curves for analyte injections.Kinetic analysis of the data was performed using 1:1 Langmuir bindingmodel with global fit. The result for each mAb was reported in theformat of K_(a) (On-rate), K_(d) (Off-rate), K_(D) (Equilibriumdissociation constant), and percent activity. The affinities of theC2177 HFR variants were similar to the parent M40 mAb, showing less thana threefold change in K_(D) for all variants. Similarly, the affinitiesof the HFR C2191 human mAb variants showed less than a two-folddifference from the parent M41 mAb.

The bioactivity of the HFR variants was measured by their inhibition ofCD70-mediated induction of NFkB in a Luciferase reporter assay. HEKcells were transfected with an NFkB inducible luciferase expressionvector pGL4-32-NFkB-Luc2 (Promega), and CD27 expression plasmid or emptyvector and incubated overnight in Freestyle expression medium (Gibco,#12338). The next day cells were plated in 96-well culture plates in 40uL, and 50,000 cells per well. Then, 40 uL antibodies or controls wereadded to cells using a serial dilution of 1:3, starting at 30 ug/mlfinal in-well concentration, and incubated for 1 to 2 hours. During thisincubation, CD70 episomal cells are prepared for stimulation. Briefly,adherent cells were resuspended using standard cell culture techniquesand incubated for 1 hour with Mitomycin C at 25 ug/mL to stop cellexpansion. After incubation, CD70+ cells were washed in medium, diluted,and 40 uL was added at 10,000 cells per well. The plates were incubatedovernight. The next day Steady Glo reagent (Promega) was prepared perthe manufacturer's instructions and 120 uL was added per well. Plateswere incubated at room temperature for 20 minutes while shaking.Luminescence was measured on a Perkin Elmer Envision Reader. The IC₅₀sof the C2177 HFR variants were similar to each other and to M40 parentalMAb, ranging from 0.11 nM to 0.21 nM. The IC₅₀s of the C2191 HFRvariants also were similar to each other and to the M41 parent, varyingfrom 0.13 nM to 1.39 nM.

Consideration of affinity, bioactivity and biophysical properties led tothe selection of the C2177 variant M69, comprised of the variableregions H28 (SEQ ID NO: 111) and L35 (SEQ ID NO: 82), and the C2191variant M91, comprised of the variable regions H31 (SEQ ID NO: 131) andL42 (SEQ ID NO: 140), for affinity maturation. A summary of K_(D)s,purification yield, binding to CD27 ECD for the cell culturesupernatants (“ELISA”), and inhibition (IC₅₀) of CD27 mediated NFκβresponse by CD70 for the M40 parent and its M69 HFR variant and for theM41 parent and its M91 variant are shown in Table 4.

TABLE 4 NFkB Proteon Yield ELISA IC50 Protein ID VH VL KD (nM) (mg)signal (nM) C2177 parent M40 H7 L18 0.96 n.d. 1.00 0.14 M69 H28 L35 0.7710.08 1.09 0.11 C2191 parent M41 H10 L20 10.3 n.d. 1.00 0.30 M91 H31 L427.9  5.64 1.09 0.28

Example 8: Optimization of C2177 HFR MAB M69

M69 has an affinity around 1 nM to human CD27 ECD and contains the sameCDRs as C2177, and the HFA parent CD27M40. Optimization of M69 involvedmultiple libraries to increase affinity and remove PTM sites introducedor identified in the process.

As described in Example 9, a parallel phage display library approach forHFR and optimization of C2177 identified diversity in the proline atposition 52a of CDR-H2. This position was not randomized in the librarydesign. The co-structure of the C2177 and C2191 Fabs with CD27 (Example5) indicates that P52a is not directly involved in antigen binding.Nevertheless, mutation at this position could alter the CDR-H2 loopconformation and enable more optimal interactions with CD27 bysurrounding residues D27. Thus, a library was designed to randomlydiversify P52a and its neighboring residues Y52, G53 and D54 using NNKmutagenesis (library C27H28L2). Also in the path 2 optimization, a Y32to F mutation in CDR-L1 showed improved binding. Thus, a second librarywas designed with random diversity in Y32 together with diversity inresidues Y30a, D30d, A50, which lie in the same structural plane as Y32(library C27L35L2).

In addition, the complete CDR-H3 and CDR-L1 loops were evaluated usinglibraries of limited diversity. Tables 5 and 6 show the design of theselibraries.

TABLE 5 Limited diversity affinity maturation design for CDR-H3(C27H28L3) VH Parent amino acid and position Diversity Ser95 A, S Asp96A, D Tyr97 A, D, S, Y Tyr98 A, D, S, Y Gly99 A, G Asp100 A, D Tyr100a A,D, S, Y Gly100b A, G Phe100c A, F, S, V Ala101 A, G Tyr102 A, D, S, Y

TABLE 6 Limited diversity affinity maturation design for CDR-L1(C27L35L3) VH Parent amino acid and position Diversity Lys24 A, K, E, TAla25 A, G Ser26 A, S Gln27 A, Q, E, P Ser28 A, S Val29 A, V Asp30 A, DTyr30a A, D, S Ala30b A, G Gly30c A, G Asp30d A, D Ser31 A, S Tyr32 A,D, S Met33 A, M, T, V Asn34 A, N, D, T

Fab libraries were constructed in a pIX phage Fab display system asdescribed in WO2009/085462, Shi et al, J Mol Biol 397: 385-396 (2010),and Tornetta et al. J Immunol Methods 360: 39-46 (2010) with minormodifications to restriction enzyme sites. These libraries were pannedagainst biotinylated CD27-ECD according to panning schemes known in theart, such as described in WO2009/085462 and in Shi et al, J Mol Biol397: 385-396 (2010), directed to increasing affinity by selecting for aslower off-rate or faster on-rate. Phage was produced by helper phageinfection. Binders were retrieved by addition of beads to form abead/antigen/phage complex. After the final wash, phage was rescued byinfection of exponentially growing TG-1 Escherichia coli cells. Phagewas again produced and subjected for additional rounds of panning.

For follow-up screening, DNA was prepared from glycerol stocks of phagepanning rounds and the pIX gene was excised by NheI/SpeI digestion.After ligation, the DNA was transformed into TG-1 cells and grown onLB/Agar plates overnight. The next day, colonies were picked, grownovernight, and the cultures used for (i) colony PCR and sequencing ofthe V-regions, and (ii) induction of Fab production. For Fab production,the overnight culture was diluted 10-100 fold in new media and grown for5-6 hours at 37 degrees C. Fab production was induced by the addition offresh media containing IPTG and the cultures were grown overnight at 30degrees C. The following day, the cultures were spun down and thesupernatants, containing the soluble Fab proteins, were used for FabELISA. For the ELISA, the soluble Fab proteins were captured onto platesby a polyclonal anti-Fd(CH1) antibody. After washing and blocking,biotinylated human CD27 ECD was added at 0.2 nM concentration. Thisconcentration enables ranking of the Fab variants, defined as percentbinding of the parent, in which the parent Fab, present as a control inall plates, is defined as 100% binding. The biotinylated CD27 ECD wasdetected by HRP-conjugated streptavidin and chemiluminescence read in aplate reader. At this concentration of CD27, ranking of the Fabvariants, normalized to the parent Fab, is possible. By this criterion,10 heavy and 6 light chains binding human CD27 at 100% or higherrelative to M69 Fab were selected.

From the CDR-H2 library (C27H28L2), the parental Y was predominantlyselected at position 52 indicating preference for this residue. Atposition 52a, P was replaced with A, S, V, and G residues among the Fabswith the best binding activity. At position 53, the parental G wasselected along with R and N. At position 54, only the parental D wasrecovered. Nine clones from this library (Table 7) were subcloned intoIgG vectors for expression and characterization as mAbs.

TABLE 7 Nine VH clones selected from full diversity library C27H28L2Peptide ID Y52 P52a G53 D54 H237 F V R D H238 Y V G D H239 Y A G D H240Y A R D H241 Y G R D H242 Y A N D H243 Y G G D H244 Y S G D H245 Y S R D

For the CDR-H3 library (C27H28L3), the only diversity recovered was S95Aand A101G. One clone from this library containing both mutations (Table8) was subcloned into the IgG vectors for expression andcharacterization as a mAb.

TABLE 8 Single VH clone selected from C27H28L3 Peptide ID S95 D96 Y97Y98 G99 D100 Y100a G100b F100c A101 Y102 H236 A D Y Y G D Y G F G Y

For the four position L-CDR1 library (C27L35L2), position 30a showedenrichment of the parental Y and W. At position 30d, residues S, H, andE were enriched along with the parental D. At position 32, the parentalY was replaced with F and W. At position 50, T was preferred over theparental A. In general, the best clones had more hydrophobic side chainscompared to parent. Five clones from this library (Table 9) weresubcloned into IgG vectors for expression and characterization as mAbs.

For the complete CDR-L1 library with limited diversity (C27L35L3), onlyone sequence was recovered, with the only difference from parent beingY32 changed to F, similar to the four position VL library above. Thiscomplete CDR-L1 library did not include an F in position 32, and thusthe recovered clone was likely a contaminant from the four position VLlibrary. This clone (L255) was subcloned into the IgG vectors forexpression and characterization as a mAb (Table 9).

TABLE 9 Six VL clones selected from C27L35L1 and C27L35L2 Peptide IDY30a D30d Y32 A50 L255 Y D F A L256 Y D W V L257 Y D W T L258 Y S F TL260 W H W T L261 Y S F E

The 6 variant light chains were paired with the 10 variant heavy chainsto give 60 combinations that were expressed HEK293E cells. Supernatantswere screened for expression level, binding to human CD27 ECD asmeasured by ELISA, and affinity as measured on a ProteOn instrument. Theexpression level of all variants was sufficient for screening purposes.Affinity was increased up to 40-fold for some variants. Two mAbs M596and M600, were selected for further mutagenesis to remove potentialsites of post-translational modification. The VH and VL chaincombinations for these mAbs are given in Table 10. The antibodies differby only two residues in their light chains.

TABLE 10 Heavy and light chain pairing of selectedC2177 affinity matured leads Light Heavy Chain CDR-L2 Chain CDR-H2Antibody Peptide (SEQ ID Peptide (SEQ ID ID ID CDR-L1 (SEQ ID NO) NO) IDNO) M596 L257 KASQSVDYAGDSWMN (26) TASNLES H239 RIYAGDGDTN (39)(residues 1-10 of 15) M600 L255 KASQSVDYAGDSFMN (25) AASNLES H239RIYAGDGDTN (37) (residues 1-10 of 15)

M596 differs from the parent molecule, M69, at three positions: P52aA inCDR-H2, Y32W in CDR-L1, and A50T in CDR-L2. M600 differs from M69 at twopositions: P52aA mutation in CDR-H2 and Y32F in CDR-L1.

Three shared potential post-translational modification sites wereidentified in M596 and M600. There is a potential N-linked glycosylationsite at position N58 in CDR-H2 and two potential isomerization sites inCDR-H2 and CDR-L1 encoded by “DG” and “DS,” respectively. In addition,M596 contains a non-germline tryptophan residue in CDR-L1 that could besusceptible to oxidation.

To remove the glycosylation risk, three individual single substitutionswere created at N58 and one at S60 (Table 11). The constructs wereexpressed in HEK293E cells and supernatants were evaluated for affinityto CD27 using the ProteOn instrument. All of the variants had affinitiesclose to those of the parents which were 25 pM and 49 pM for M596 andM600, respectively. Variants M680 and M678, both derived from M600, wereselected for evaluation of further substitutions to eliminate theisomerization sites. Variants M680 and M678 have A at positions 60 and58, respectively, and have the additional advantage of lacking thetryptophan in CDR-L1 that was present in the M596 parent.

TABLE 11 VH Parent amino acid and position Diversity Asn58 N, A, R, TSer60 S, A

To evaluate the impact of mutating the potential isomerization sites inM678 and M680, a small library was designed to remove both sites inparallel. Each mutation was substituted individually into CDR-H2 of theheavy chains or CDR-L1 of the common light chain and then paired in acombinatorial library. The diversity of this library is shown in Table12.

TABLE 12 Diversity VH Parent amino acid and position Asp54 D, E Gly55 G,A VL Parent amino acid and position Asp34 D, E

These mAbs were expressed and affinity was evaluated as for theglycosylation site variants. The D34E mutation in the CDR-L1 potentialisomerization site led to a consistent two-fold increase in affinity andtherefore this site was successfully removed. The D54E mutation in theCDR-H2 potential isomerization site lowered the affinity more thantenfold. However, the G55A mutation did not significantly affect theaffinity. Variants M703 and M706 retain the affinity of the M600 parentand have a reduced risk of impact on function from PTM. Table 13 showsthe selected variants from each stage of the PTM-risk assessment, theirheavy and light chain pairing, affinity, and sequence modifications inthe CDRs. The mutation selected to remove the potential glycosylationsite is underlined. The mutations to remove the two potentialisomerization sites are bolded and double underlined.

TABLE 13 mAb ID VH VL KD (pM) CDR-H2 (SEQ ID NO) CDR-L1 (SEQ ID NO) M596H239 L257 25 RIYAGDGDTNYSPSFQG KASQSVDYAGDSWMN (165) (26) M600 H239 L25549 RIYAGDGDTNYSPSFQG KASQSVDYAGDSFMN (165) (25) M678 H259 L255 53RIYAGDGDTAYSPSFQG KASQSVDYAGDSFMN (166) (25) M680 H260 L255 30RIYAGDGDTNYAPSFQG KASQSVDYAGDSFMN (167) (25) M703 H270 L267 28 RIYAGD ADTAYSPSFQG KASQSVDYAG E SFMN (168) (29) M706 H272 L267 13 RIYAGD ADTNYAPSFQG KASQSVDYAG E SFMN (169) (29)

Example 9: Combinded HFR and Optimization of C2177 MAB

In this approach, a limited set of HFR variants were evaluated in a Fabformat for expression, pIX display and binding and the best candidateswere then advanced into optimization. CDRs from C2177 were humanframework adapted into two heavy chains VH5-51 (SEQ ID NO: 102 H24) andVH1-46 (SEQ ID NO: 106 H25) and two light chains Vk4-1 and Vk012 (SEQ IDNO: 90 L36). These HFA variable domains were paired together in a 2×2matrix as Fabs with human CH1 and Ck constant regions in the Fab pIXphage display vector. The VH1-46/Vk012 variant (M55, H25/L36) showedbinding to CD27 and good display characteristics and was selected forconstruction of affinity maturation libraries.

The Fab libraries for pIX phage display were constructed as describedabove for Example 8. Based on the experimental co-structure of CD27 withC2191 and C2177 (Example 5), diversity libraries were designed in CDRresidues in and around the antibody paratope. The emphasis on variationwas in CDRs L1, L3, and H2. A total of 4-6 residues within an individualCDR were diversified with an NNK codon, encoding for all 20 amino acids.The size of each library was estimated to ≤6×10⁷ variants, which can becovered using standard library restriction endonuclease cloningtechniques. Table 14 shows the residues that were subjected to fulldiversification in the different CDR libraries.

TABLE 14 Affinity maturation library design for C2177 Parent amino acidand position VH CDR CDR-H2 Y52 G53 D54 D56 N58 CDR-H3 Y97 Y98 D100 Y100aVL CDR CDR-L1 Y30a A30b G30c D30d Y32 CDR-L3 Q90 N92 E93 D94 Y96

Fab libraries displayed on phage coat protein IX were panned againstbiotinylated hCD27ECD/Fc. Phage was produced by helper phage infectionof a plasmid library of the variants. Binders were retrieved by additionof streptavidin-coated magnetic beads to form a bead/antigen/phagecomplex. After the final wash, phage was rescued by infection ofexponentially growing MC1061F′ Escherichia coli cells. Phage was againproduced and subjected for additional rounds of panning. Soluble Fabfrom selected clones was produced and evaluated for binding activity asdescribed about for Path 1. Hits were obtained only from the CDR-L1 andCDR-L2 libraries. Twenty-one clones from these two librariesdemonstrated binding greater than that of the parent HFR Fab. Clonescontaining C or M in the diversified sequences were discarded. Ten Fabswere converted for expression in a IgG4SPAAa/kappa background forfurther characterization. The IgG4PAA heavy chain is human IgG4containing a serine to proline substitution in the hinge region (Angalet al., Mol Immunol 30: 105 (1993) and alanine substitutions at twopositions in CH2 (M L Alegre et al, Transplantation; 57: 1537-43(1994)). The mAbs were produced in HEK293E cells as replicas of the Fabsand as a matrix of heavy and light chain combinations. Affinity wasmeasured on a ProteOn instrument using the culture supernatants (Table15). Mutation of P52a to Q (M158) or S (M157) in CDR-H2 decreased theK_(D) 6-fold compared to that of the parental mAb (M159). The Y36Fmutation in CDR-L1 (M149) decreased K_(D) 4-fold and the addition ofG33H and D34E mutations (M155) led to a 6-fold decrease in K_(D).Combination of the P52S mutation with either Y36F (M160) or Y36F plusG33H and D34E decreased the K_(D) 20-fold to 100 pM. The combinations ofsubstitutions in M158, M160 and M166 were selected for furthercharacterization.

TABLE 15 Initial panel of mAbs derived from Fab maturation librariesProtein DNA ID H&L CDR-H2 (SEQ ID NO) CDR-L1 (SEQ ID NO) K_(D )(nM) M149H25, L219 RIYPGDGDTNYNGKFKG KASQSVDYAGDSFMN 0.54 (3) (25) M150 H25, L218RIYPGDGDTNYNGKFKG KASQSVDYFGDSLMN 4.04 (3) (32) M151 H25, L224RIYPGDGDTNYNGKFKG KASQSVDYYNSSFMN 1.07 (3) (36) M152 H25, L223RIYPGDGDTNYNGKFKG KASQSVDYWSDSFMN 1.54 (3) (35) M153 H25, L222RIYPGDGDTNYNGKFKG KASQSVDYVGTSFMN 1.41 (3) (34) M154 H25, L221RIYPGDGDTNYNGKFKG KASQSVDYFRTSFMN 1.56 (3) (33) M155 H25, L217RIYPGDGDTNYNGKFKG KASQSVDYAHESFMN 0.37 (3) (31) M156 H25, L216RIYPGDGDTNYNGKFKG KASQSVDYFSESFMN 0.71 (3) (170) M157 H197, L220RIYQGDGDTNYNGKFKG KASQSVDYAGDSYMN 0.39 (22) (24) M158 H196, L220RIYSGDGDTNYNGKFKG KASQSVDYAGDSYMN 0.36 (19) (24) M159 H25, L220RIYPGDGDTNYNGKFKG KASQSVDYAGDSYMN 2.23 (3) (24) M160 H196, L219RIYSGDGDTNYNGKFKG KASQSVDYAGDSFMN 0.12 (19) (25) M161 H196, L218RIYSGDGDTNYNGKFKG KASQSVDYFGDSLMN 1.34 (19) (32) M162 H196, L224RIYSGDGDTNYNGKFKG KASQSVDYYNSSFMN 0.43 (19) (36) M163 H196, L223RIYSGDGDTNYNGKFKG KASQSVDYWSDSBFMN 0.30 (19) (35) M164 H196, L222RIYSGDGDTNYNGKFKG KASQSVDYVGTSFMN 0.27 (19) (34) M165 H196, L221RIYSGDGDTNYNGKFKG KASQSVDYFRTSFMN 0.18 (19) (33) M166 H196, L217RIYSGDGDTNYNGKFKG KASQSVDYAHESFMN 0.10 (19) (31) M167 H196, L216RIYSGDGDTNYNGKFKG KASQSVDYFSESFMN 0.91 (19) (170)

M158, M160 and M166 proteins were produced in 750 mL cultures of HEK293cells, purified, and analyzed for binding kinetics to CD27-His onBiacore. The K_(D) values were about 1 log higher than those measured byProteOn with crude supernatants but showed the same values relative toeach other (Table 16).

TABLE 16 mAb ID k_(on) (M⁻¹s⁻¹) k_(off) (s⁻¹) K_(D) (pM) M158 (1.5 ±0.03)E+06   (6.6 ± 1.7)E−04 439 ± 114 M160 (1.6 ± 0.15)E+06 ((1.8 ±0.01)E−04 117 ± 11  M166 (1.62 ± 0.04)E+06  (2.03 ± 0.16)E−04 126 ± 11 

When re-evaluating the original HFA combinations, the VH5-51 adapted VHshowed a 2-fold lower K_(D) than the VH1-46 scaffold. The P52aS mutationin H-CDR2 was introduced into the VH5-51 VH creating H221. H221 wasexpressed with the L220, L219 and L217 light chains from the HFAparental mAb (M159) and the affinity improved variants M160 and M166,respectively, to generate mAbs M171, M169 and M170. BIAcore kineticmeasurements on the purified mAbs showed a two-fold improvement in K_(D)in comparison to the corresponding VH1-46 variants (compare Tables 16and 17).

TABLE 17 H/L K_(D) Ave mAb ID Peptide ID K_(on) Ave (M⁻¹s−¹) K_(off) Ave(s⁻¹) (pM) M169 H221, L219 (1.48 ± 0.13)E+06 1.02 ± 0.07)E−04 69 ± 8M170 H221, L217 1.66E+06 1.16E−04 70 M171 H221, L220 1.53E+06 3.58E−04234

Sequence analysis of M160, M169 and M170 identified a potentialisomerization site at D54-G55 and a potential deamidation site atN61-G62 in CDR-H2 of H196 and H221. Additionally, a potentialisomerization site was identified at D34 within CDR-L1 of L219.Mutations were introduced to remove these sites and evaluated for theirimpact on activity (Table 18). Purified mAbs were analyzed for affinityto CD27-His on ProteOn. The mutations either had no effect or a positiveeffect on K_(D). For example, both M668 and M671 had almost 2 fold lowerK_(D)s than their parental mAbs, M160 and CM169, respectively.

TABLE 18 mAb variants with PTM sequences mutated K_(D) Parent mAb H/Lk_(on )Ave k_(off )Ave Ave mAb ID Peptide ID CDR-H2 (SEQ ID NO)CDR-L1 (SEQ ID NO) (M⁻¹s⁻¹) (s⁻¹) (pM) M160 M160 H196, L219RIYSGDGDTNYNGKFKG KASQSVDYAGDSFMN 2.07E+06 2.23E−04 108 (19) (25) M166M166 H196, L217 RIYSGDGDTNYNGKFKG KASQSVDYAHESFMN 1.84E+06 2.34E−04 127(19) (31) M169 M169 H221, L219 RIYSGDGDTNYNGKFKG KASQSVDYAGDSFMN2.36E+06 1.39E−04 59 (19) (25) M160 M668 H255, L266 RIYSGDADTNYAQKFKGKASQSVDYAGESFMN 2.14E+06 1.29E−04 60 (20) (29) M160 M669 H256, L266RIYSGDADTNYNQKFKG KASQSVDYAGESFMN 2.49E+06 1.45E−04 58 (21) (29) M169M670 H257, L266 RIYSGDADTNYAQKFKG KASQSVDYAGESFMN 1.73E+06 1.13E−04 65(20) (29) M169 M671 H258, L266 RIYSGDADTNYNQKFKG KASQSVDYAGESFMN2.52E+06 9.73E−05 39 (21) (29) M166 M672 H255, L217 RIYSGDADTNYAQKFKGKASQSVDYAGESFMN 1.83E+06 2.64E−04 144 (20) (29) M166 M673 H256, L217RIYSGDADTNYNQKFKG KASQSVDYAGESFMN 1.89E+06 1.89E−04 100 (21) (29)

Example 10: Optimization of C2191 HFR MAB M91

The methods applied for the optimization of the M91 (H31/L42) were asdescribed in Example 8, except as noted. An alanine/germline scan of theCDRs of C2191 was carried out in a Fab format to evaluate positionsimportant for interaction with CD27, using the C2191 parent VH and VLregions in a Fab format with human Ch1 and Ck constant regions. Thelibraries replaced the residues in the CDRs with alanine or the residuepresent in the corresponding germline sequence. Some positions in theCDRs were excluded as they had low or no solvent exposure based onmodeling and subsequently on the determined structure (Example 5).Putative somatic mutations were back-mutated to mouse germline aminoacids to assess their contribution to antibody affinity. Briefly, themouse V regions were cloned into the Fab pIX display vector and thebinding of the parent to biotinylated human CD27-ECD protein wasverified by ELISA. Single mutations (according to the library design)were introduced by site-directed mutagenesis, performed essentially asdescribed by Stratagene (La Jolla, Calif., USA). Sequence confirmedmutants were cherry-picked into new plates and grown together withparental Fab and negative control Fabs. The final singleamino-acid-substitution variants were generated in E. coli, and thenscreened for expression and CD27 binding by ELISA. The expression andbinding signals for the parent clones were averaged and set to 1.0 andthe signals of the mutants were normalized relative to the parent. Twoforms of antigen were used in the ELISAs: CD27 ECD (1-173 residues) andCD27 ECD-Fc chimera (R&D Systems).

The results of this scan coupled with co-crystal structure provided thebasis for design of affinity maturation libraries. For the heavy chain;the positions selected for variation were T33 in H-CDR1 and Y50, S52,S52a, N56 and Y58 in CDR-H2 (Table 23). T33 is not a contact residue buta T33A mutation improved binding. Positions S52 and S52a are not contactresidues but the substitutions in the scan showed some increasedbinding. The tyrosines at positions 50 and 58 are both contact sites andsubstitutions at these sites were selected in the parallel optimizationpath described in Example 11. Position N56 was not evaluated in thealanine/germline scan but it is a contact site and adjacent to T33, S52,and S52a in the crystal structure. For the light chain, the positionsselected for variation were T30a, S30b, G30c and Y30d in CDR-L1 and L50and N53 in CDR-L2 (Table 23). None of these residues contact antigendirectly but are adjacent to residues that are in contact which the scanshowed had substantial negative impact on binding. A L50A mutation had amoderate effect on binding and, in the crystal structure, is the onlyresidue in CDR-L2 likely in contact with antigen. In addition, N53 wasselected for limited diversification. Two parallel libraries wereconstructed, one with Y30d mutated to W, and another with Y30d kept asY, since W could make the paratope more hydrophobic and thus lessdevelopable. Tables 19 and 20 below show the VH and VL affinitymaturation library designs for M91.

TABLE 19 2191 HC_CDR1 HC_CDR2 Antigen No Yes No No Yes Yes contact?Position in HC T33 Y50 S52 S52a N56 Y58 (SEQ ID NO: 131) Position in HCT33 Y50 S52 S53 N57 Y59 (SEQ ID NO: 131) Sequence All 20 Y All 20 All 20All 20 Y Diversity amino A amino amino amino I acids W acids acids acidsL H W

TABLE 20 2191 LC_CDR1 LC_CDR2 Antigen contact No No No Yes Yes NoPosition in LC T30a S30b G30c Y30d L50 N53 (SEQ ID NO: 140) Position inLC T31 S32 G33 Y34 L54 N57 (SEQ ID NO: 140) Sequence Diversity All All20 G Y* All 20 N 20 amino R W* amino K amino acids acids R acids *Twoseparate libraries containing either Y or W at this position werecreated

Fab libraries displayed on phage coat protein IX were panned againstbiotinylated CD27-ECD. A total of 12 heavy and 12 light chain variantswere selected that bound to CD27 equally or better than the parentalchimeric Fab of C2191. The variants were converted to IgG1/kappaantibodies, produced in HEK293E cells as 144 combinations, and culturesupernatants were evaluated for binding by ProteOn. Significantincreases (>100-fold) in affinity were observed for some variants. Ofthe 144 VH and VL pairings, 8 were selected for further characterization(Table 21). These mAbs were classified into three sub-groups: Group 1variants have the same heavy chain (H227, SEQ ID NO: 133) paired withfour different light chains, while Group 2 and Group 3 each have onelight chain paired with two different heavy chains. The four selectedlight chains varied at all four positions diversified in CDR-L1(RASKSVSX₁X₂X₃X₄SFMH) (SEQ ID NO: 158); where X₁ is A, E, H, or L; X₂ isD, G, V, or W; X₃ is G or R; and X₄ is W or Y). They also varied in bothpositions diversified in CDR-L2 (X₁ASX₂LES) (SEQ ID NO:171); where X₁ isL or V; and where X₂ is K, N, or R). CDR-L3 was unaltered from the L42sequence (SEQ ID NO: 140) and is QHSRELPWT.

TABLE 21Pairing of heavy and light chain sequences of selected 2191 affinity matured leadsLight Chain CDR-L2 Heavy Antibody Peptide (SEQ ID Chain CDR-H1 (SEQ IDID CDR-L1 (SEQ ID NO:) NO:) Peptide ID ID NO:) CDR-H2 (SEQ ID NO:) M427C27L244 RASKSVSAWGYSFMH VASRLES C27H227 GFTFSSYGMS YIDEGGGQTIYPDSVKG(60) (68) (44) (47) M429 C27L245 RASKSVSHVRWSFMH LASKLES C27H227GFTFSSYGMS YIDEGGGQTIYPDSVKG (61) (69) (44) (47) M488 C27L249RASKSVSEGRWSFMH VASRLES C27H227 GFTFSSYGMS YIDEGGGQTIYPDSVKG (62) (68)(44) (47) M489 C27L250 RASKSVSLDRWSFMH LASNLES C27H227 GFTFSSYGMSYIDEGGGQTIYPDSVKG (63) (67) (44) (47) M492 C27L249 RASKSVSEGRWSFMHVASRLES C27H228 GFTFSSYSMS YIDAGGGFTIYPDSVKG (62) (68) (45) (48) M493C27L250 RASKSVSLDRWSFMH LASNLES C27H228 GFTFSSYSMS YIDAGGGFTIYPDSVKG(63) (67) (45) (48) M501 C27L250 RASKSVSLDRWSFMH LASNLES C27H231GFTFSSYSMS HIDAGGGRTWYPDSVKG (63) (67) (45) (49) M526 C27L249RASKSVSEGRWSFMH VASRLES C27H222 GFTFSSYGMS YIDRGGGVTIYPDSVKG (62) (68)(44) (50)

These eight variants were produced by transient expression in HEK293Ecells in a volume of 750 ml. The harvested supernatants were purifiedvia Protein A chromatography, and each variant was analyzed by SDS-PAGEand SE-HPLC to determine purity of the sample and percentage of monomerin the purified sample. All of the variants were greater than 90% pureand greater than 90% monomeric. To evaluate association properties ofthe antibodies, retention factors (k′) were determined by performingcross-interaction chromatography for each purified variant (Jacobs S A,Wu S J, Feng Y, Bethea D & O'Neil K T (2010) Cross-interactionchromatography: a rapid method to identify highly soluble monoclonalantibody candidates. Pharm Res 27, 65-71). In this method, sampleantibodies were passed through a column coupled with human IgG andevaluated for retention relative to control antibodies. Briefly, 50 mgof human IgG (Sigma Aldrich) were coupled to a 1 mL NHS-Sepharose column(GE Healthcare) following the manufacturer's instructions. Uncoupled IgGwas removed by washing with 0.1M Tris, pH 8, 0.5M NaCl and unreacted NHSgroups were blocked with the same buffer. The coupling efficiency wasdetermined by measuring the protein concentration remaining in theunreacted coupling buffer and washes using Pierce's Coomassie Plus AssayKit (Thermo Pierce) and subtracting from the amount of protein beforeimmobilization. A control column was also prepared using the sameprotocol but without conjugation of IgG to the resin. The control columnwas run first on a Dionex UltiMate 3000 HPLC after being equilibratedwith PBS, pH Tat a flow rate of 0.1 mL/min. 20 μL of the stock proteinsolution was injected first to ensure non-specific binding sites wereblocked followed by 20 μL of 10% acetone to check the integrity of thecolumn. Samples to be analyzed were diluted to 0.1 mg/mL in PBS, pH 7.20 uL of each sample was injected onto each column and allowed to run at0.1 mL/min for 30 min. Retention times were recorded and the retentionfactor (k′) was calculated for each variant. The k′ value was calculatedas the difference in the retention times on the IgG and blank columns.All of the variants were purified to greater than 90% purity based onSDS-PAGE and SE-HPLC. All k′ values were calculated to be less than 0.3,indicative of good solution properties (Table 22).

TABLE 22 Batch analysis of purified affinity matured variants Conc.Total % Antibody ID HC LC (mg/ml) Protein Monomer Gel k′ M427 H227 L2441.42 15.63 100 ok 0.02 M429 H227 L245 0.97 10.21 100 ok 0.07 M488 H227L249 2.00 27.06 98.9 ok 0.07 M489 H227 L250 1.59 23.03 100 ok 0.17 M492H228 L249 2.07 27.96 97.4 ok 0.25 M493 H228 L250 0.58 8.12 100 ok 0.24M501 H231 L250 2.01 26.08 100 ok 0.28 M526 H222 L249 0.90 10.74 100 ok0.10

The eight variant mAbs and the HFR parent were evaluated for theiraffinity to CD27 ECD by BIAcore and their IC₅₀ in the κβ-reporter assay.Kinetic constants and affinity were measured by BIAcore. Table 23summarizes the data collected on these variants. The expression andELISA signal for binding to CD27 as measured from the initial smallculture supernatants are also included in this table.

TABLE 23 C2191 AM library subset data summary NFκβ Protein ExpressionELISA IC₅₀ K_(D) ID (ug/ml signal (pM) k_(a) k_(d) (pM) M41 n.d. n.d. 456.20E+05 8.44E−03 13650 M427 14.6 0.93 19 7.18E+05 3.00E−05 41.7 M42916.1 0.86 42 6.62E+05 2.02E−05 30.4 M488 20.7 0.92 23 1.01E+06 3.96E−0539.4 M489 24.2 0.96 41 9.06E+05 2.18E−05 24.1 M492 20.1 0.77 15 1.03E+061.47E−04 142.0 M493 24.5 0.85 14 7.23E+05 1.05E−04 145.0 M501 30.9 0.774 8.31E+05 6.16E−05 74.2 M526 14.3 0.69 6 1.00E+06 1.71E−04 171.0

Example 11: Combinded HFR and Optimization of C2191 MAB

In this approach, a limited set of HFR variants were evaluated in a Fabformat for expression, pIX display and binding and the best candidateswere then advanced into optimization. CDRs from C2191 were humanframework adapted into two heavy chains (VH3-23 and VH3-11) and twolight chains (Vk4-1 and Vk012). These HFA variable domains were pairedtogether in a 2×2 matrix as Fabs with human CH1 and Ck constant regionsin the Fab pIX phage display vector. The VH3-23Nk012 variant (H39 (SEQID NO: 145)) and L40 (SEQ ID NO: 137) showed binding to CD27 and gooddisplay characteristics and was selected for construction of affinitymaturation libraries. This Fab is referred to as “parent.”

For selection of antibodies with improved affinity, multiple residues inall CDRs except CDR-H3 of H39 were fully diversified using NNKdegenerate codons (Table 24). Each CDR library was constructedseparately and subjected to phage panning for selection of affinitymatured variants.

TABLE 24 Affinity maturation library design for C2191 variants Parentamino acid and position VH CDR CDR-H2 Y50 S52 S53 N56 Y58 CDR-H3 H95 R96G97 N98 P99 VL CDR CDR-L1 T30a S30b G30c Y30d F32 CDR-L2 L50 A51 S52 N53L54 E55 S56 CDR-L3 H90 R92 E93 L94 Y96The C2191 libraries with diversity in CDR-H2, CDR-L1 or CDR-L2 yielded50 unique Fabs with improved binding to human CD27 relative to theparental HFR Fab as measured in the single point ELISA. These cloneswere further ranked in a multi-point ELISA and seventeen clones wereselected for conversion to human IgG4alaala/kappa for furthercharacterization (Table 25).

TABLE 25 Affinity matured Fabs selected for conversion to IgG HC LCCDR-L2 Peptide Peptide CDR-H2 (SEQ ID CDR-L1 (SEQ ID (SEQ ID Fab ID IDID NO:) NO:) NO:) Parent H39 L40 YISSGGGNTYYPDSVKG RASKSVSTSGYSFMHLASNLES (46) (59) (67) F116 H39 L59 YISSGGGNTYYPDSVKG RASKSVSTSGYSFMHVGNRLED (46) (59) (70) F119 H39 L62 YISSGGGNTYYPDSVKG RASKSVSTSGYSFMHVGDRRQE (46) (59) (71) F178 H145 L40 YISGGGGQTLYPDSVKG RASKSVSTSGYSFMHLASNLES (54) (59) (67) F18 H40 L40 AIDHGGGRTYYPDSVKG RASKSVSTSGYSFMHLASNLES (51) (59) (67) F19 H41 L40 AIDHGGGRTWYPDSVKG RASKSVSTSGYSFMHLASNLES (52) (59) (67) F243 H39 L124 YISSGGGNTYYPDSVKG RASKSVSTSGYSFMHVGSRMAF (46) (59) (72) F250 H39 L131 YISSGGGNTYYPDSVKG RASKSVSTSGYSFMHVGDRANW (46) (59) (73) F256 H39 L137 YISSGGGNTYYPDSVKG RASKSVSTSGYSFMHVGSRLDY (46) (59) (74) F279 H39 L160 YISSGGGNTYYPDSVKG RASKSVSYVRWSFMHLASNLES (46) (64) (67) F291 H39 L172 YISSGGGNTYYPDSVKG RASKSVSHIRWSFMHLASNLES (46) (65) (67) F292 H39 L173 YISSGGGNTYYPDSVKG RASKSVSHVRWSFMHLASNLES (46) (61) (67) F295 H39 L176 YISSGGGNTYYPDSVKG RASKSVSTSGYSFMHVADRVEV (46) (59) (173) F297 H190 L40 TIDRGGGSTWYPDSVKG RASKSVSTSGYSFMHLASNLES (55) (59) (67) F298 H191 L40 AIDGGGGATYYPDSVKG RASKSVSTSGYSFMHLASNLES (56) (59) (67) F299 H192 L40 VIDHGGGSTHYPDSVKG RASKSVSTSGYSFMHLASNLES (57) (59) (67) F302 H39 L179 YISSGGGNTYYPDSVKG RASKSVSLIRWSFMHLASNLES (46) (172) (67) F57 H79 L40 AIDHGGGQTLYPDSVKG RASKSVSTSGYSFMHLASNLES (53) (59) (67)

IgG1/k mAbs were constructed as replicas of the Fabs and as a matrix ofheavy and light chain variable regions (Table 26) and tested foraffinity and solution properties. The mAb form of the parent Fab isdenoted M131. M141 and M408 were selected for further characterization.

TABLE 26 mAbs derived from Fab affinity maturation mAb Fab ID ID H & LPeptide ID Parent M131 H39, L40 F18 M132 H40, L40 F57 M133 H79, L40 F298M134 H191, L40 F299 M135 H192, L40 F292 M136 H39, L173 F279 M137 H39,L160 F256 M138 H39, L137 Combination M139 C27H40, L173 Combination M140C27H40, L160 Combination M141 C27H79, L173 Combination M142 C27H79, L160Combination M143 C27H191, L160 Combination M144 C27H191, L173Combination M145 C27H192, L173 Combination M146 H192, L160 CombinationM408 H192, L137

Example 12: Characterization of Affinity Matured MABS

Selected affinity matured mAbs derived from the parental C2177 and C2191hybridoma antibodies were codon optimized, introduced into a differentvector for dual-expression of heavy and light chains, expressed inCHO-GS cell culture, and purified for further characterization. The IDsof these antibodies in relation to the matured variants described in theExamples above are shown in Table 27.

TABLE 27 Single- gene LC Peptide Parent DNA ID DNA ID ID HC Peptide ID2191 429 696 27L245 27H227 2191 492 695 27L249 27H228 2191 488 69427L249 27H227 2191 141 707 27H79 27L173 2191 408 708 27H192 27L137 2177703 709 27H270 27L267 2177 706 710 27H272 27L267 2177 671 711 27H25827L266 2177 668 713 27H255 27L266

Summary data for these mAbs is shown below for the K_(D) analysis byBiacore and the IC₅₀ measured in a NF-κβ reporter gene assay (Tables 28and 29). For this NF-κβ reporter assay, HEK-293F cells were transfectedwith a total of 36 ng of DNA containing both human CD27 and luciferaseconstructs, under control of the NF-κβ promoter. HEK-293F transfectantswere plated 5×10⁴ cells per well in 40 μL Freestyle media (Gibco) in96-well plates. Dilutions of anti-CD27 hybridomas mAbs were added to theassay plate in Freestyle media for a final concentration of 50 μg/mLwith 1:3 dilutions and plates were incubated at 37° C. (5% CO₂) for onehour. To test for ability of mAbs to neutralize CD70:CD27 signaling,terminally irradiated (4000 rads) HEK-293E CD70 episomal cells wereadded at 20% of the number of CD27 transfectant cells to the assayplate. To test for agonist activity of hybridoma mAbs, addition of CD70episomal cells was omitted. Assay plates were incubated overnight at 37°C. (5% CO₂) and developed using the Steady-Glo® Luciferase Assay System(Promega) according to the instructions of the manufacturer.

TABLE 28 Characterization of matured variants derived from C2191 NF-κβAssay mAb k_(a) (1/Ms) k_(d) (1/s) K_(D) (pM) IC₅₀ (pM) C2191 parent4.35E+05 0.0103 23678 2300 chimera (M41) M694 4.80E+05 7.60E−05 105 420M695 5.10E+05 1.70E−04 202 250 M696 3.70E+05  2.1E−05 57 330 M7075.85E+05 1.24E−04 213 260 M708 6.430E+05  2.25E−04 350 260

TABLE 29 Characterization of matured variants derived from C2177 K_(D)NF-κβ Assay mAb k_(a) (1/Ms) k_(d) (1/s) (pM) IC₅₀ (pM) C2177 parentchimera 1.06E+06 1.32E−03 1240 466 (M40) M709 2.30E+06 5.89E−05 26 272M710 2.55E+06 4.88E−05 19 320 M711 1.97E+05 5.82E−05 30 258 M7132.06E+05 1.16E−05 56 296CDR and V Region Sequences

TABLE 30 2177 path 1 CDR-H1 (SEQ CDR-H3 (SEQ VH ID NO:)CDR-H2 (SEQ ID NO:) ID NO:) Kabat Kabat Kabat H7 M40 parent SSWMN (1)RIYPGDGDTNYNGKFKG (3) SDYYGDYGFAY (23) Kabat-7 (+2 HFR Extended CDR-residues to show PMT H1 mutations) Kabat H28 HFR(M69) GYAFSSSWMNRIYPGDGDTNYS (4) SDYYGDYGFAY (2) (23) H236 GYAFSSSWMN (2) RIYPGDGDTNYSADYYGDYGFGY (162) H237 GYAFSSSWMN (2) RIFVRDGDTNYS (5) SDYYGDYGFAY (23)H238 GYAFSSSWMN (2) RIYVGDGDTNYS (6) SDYYGDYGFAY (23) H239 M596, M600GYAFSSSWMN (2) RIYAGDGDTNYS (7) SDYYGDYGFAY (23) H240 GYAFSSSWMN (2)RIYARDGDTNYS (8) SDYYGDYGFAY (23) H241 GYAFSSSWMN (2) RIYGRDGDTNYS (9)SDYYGDYGFAY (23) H242 GYAFSSSWMN (2) RIYANDGDTNYS (10) SDYYGDYGFAY (23)H243 GYAFSSSWMN (2) RIYGGDGDTNYS (11) SDYYGDYGFAY (23) H244GYAFSSSWMN (2) RIYSGDGDTNYS (12) SDYYGDYGFAY (23) H245 GYAFSSSWMN (2)RIYSRDGDTNYS (13) SDYYGDYGFAY (23) H259 M678 GYAFSSSWMN (2)RIYAGDGDTAYS (14) SDYYGDYGFAY (23) H260 M680 GYAFSSSWMN (2)RIYAGDGDTNYA (15) SDYYGDYGFAY (23) H270 M703 = M709 GYAFSSSWMN (2)RIYAGDADTAYS (16) SDYYGDYGFAY (23) H272 M706 = M710 GYAFSSSWMN (2)RIYAGDADTNYA (17) SDYYGDYGFAY (23) RIX₁X₂X₃DX₄DTX₅YX₆ (151) For SEQ IDNO: 151, X₁ is F or Y; X₂ is A, G, S, OR V; X₃ is G, N, or R; X₄ is A orG; X₅ is A or N; and X₆ is A or S

TABLE 31 2177 path 2 CDR-H1 (SEQ ID CDR-H3 (SEQ VH NO:)CDR-H2 (SEQ ID NO:) ID NO:) Kabat Kabat Kabat H7 M40 parent SSWMN (1)RIYPGDGDTNYNGKFKG (3) SDYYGDYGFAY (23) Kabat-7 (the rest ofExtended CDR- CDR is the same in H1 mouse and HFR) Kabat H24 HFR(M50)GYAFSSSWMN (2) RIYPGDGDTNYNGKFKG (18) SDYYGDYGFAY (23) H221 M169, M170,GYAFSSSWMN (2) RIYSGDGDTNYNGKFKG (19) SDYYGDYGFAY M171 (23) H257 M670GYAFSSSWMN (2) RIYSGDADTNYAQKFKG (20) SDYYGDYGFAY (23) H258 M671 = M711GYAFSSSWMN (2) RIYSGDADTNYNQKFKG (21) SDYYGDYGFAY (23) H25 HFR (M55);GYAFSSSWMN (2) RIYPGDGDTNYNGKFKG (18) SDYYGDYGFAY M149-156; (23) M159H196 M158; M160-167 GYAFSSSWMN (2) RIYSGDGDTNYNGKFKG (19) SDYYGDYGFAY(23) H255 M668 = M713; GYAFSSSWMN (2) RIYSGDADTNYAQKFKG (20) SDYYGDYGFAYM672; (23) H256 M669; M673 GYAFSSSWMN (2) RIYSGDADTNYNQKFKG (21)SDYYGDYGFAY (23) H197 M157 GYAFSSSWMN (2) RIYQGDGDTNYNGKFKG (22)SDYYGDYGFAY (23) RIYX₁GDX₂DTNYX₃X₄KFKG (152) For SEQ ID NO: 152, X₁ isP, Q, or S; X₂ is A or G; X₃ is A or N; X₄ is G or Q.

TABLE 32 2177 path 1 CDR-L1 CDR-L2 CDR-L3 (SEQ ID NO:) (SEQ ID NO:)(SEQ ID NO:) VL Kabat Kabat Kabat L18 M40 parent KASQSVDYAGDSYMNAASNLES (37) QQSNEDPYT mouse (24) (41) L35 HFR (M69) KASQSVDYAGDSYMNAASNLES (37) QQSNEDPYT (24) (41) L255 M600; M678; KASQSVDYAGDSFMNAASNLES (37) QQSNEDPYT M680 (25) (41) L256 KASQSVDYAGDSWMN VASNLES (38)QQSNEDPYT (26) (41) L257 M596 KASQSVDYAGDSWMN TASNLES (39) QQSNEDPYT(26) (41) L258 KASQSVDYAGSSFMN TASNLES (39) QQSNEDPYT (27) (41) L260KASQSVDWAGHSWMN TASNLES (39) QQSNEDPYT (28) (41) L261 KASQSVDYAGSSFMNEASNLES (40) QQSNEDPYT (27) (41) L267 M703 = M709; KASQSVDYAGESFMNAASNLES (37) QQSNEDPYT M706 = M710 (29) (41) KASQSVDX₁AGX₂SX₃X₁ASNLES (154) MN (153) For SEQ ID NO: 153, X₁ is W or Y; X₂ is D, E, Sor H; X₃ is F, W, or Y. For SEQ ID NO: 154, X₁ is A, E, T, or V;

TABLE 33 2177 path 2 CDR-L1 CDR-L2 CDR-L3 (SEQ ID NO:) (SEQ ID NO:)(SEQ ID NO:) VL Kabat Kabat Kabat L18 M40 parent KASQSVDYAGDSYMN (24)AASNLES QQSNEDPYT (41) mouse (37) L36 HFR (M55; KASQSVDYAGDSYMN (24)AASNLES QQSNEDPYT (41) M50) (37) L216 M156; M167 KASQSVDYFSESYMN (30)AASNLES QQSNEDPYT (41) (37) L217 M155; M166; KASQSVDYAHESFMN (31)AASNLES QQSNEDPYT (41) M170; (37) M672; M673 L218 M150; M161KASQSVDYFGDSLMN (32) AASNLES QQSNEDPYT (41) (37) L219 M149; M160;KASQSVDYAGDSFMN (25) AASNLES QQSNEDPYT (41) M169 (37) L266 M668 = M713;KASQSVDYAGESFMN (31) AASNLES QQSNEDPYT (41) M669; M670; (37) M671 =M711; L220 M157; M171; KASQSVDYAGDSYMN (24) AASNLES QQSNEDPYT (41)M158; M159 (37) L221 M154 KASQSVDYFRTSFMN (33) AASNLES QQSNEDPYT (41)(37) L222 M153 KASQSVDYVGTSFMN (34) AASNLES QQSNEDPYT (41) (37) L223M152; M163 KASQSVDYWSDSFMN (35) AASNLES QQSNEDPYT (41) (37) L224M151; M162 KASQSVDYYNSSFMN (36) AASNLES QQSNEDPYT (41) (37)KASQSVDYX₁X₂X₃MSX₄MN (155) For SEQ ID NO: 155, X₁ is A, F, V, W or Y; X₂is G, H, N, R, or S; X₃ is D, E, S, or T; X₄ is F, L, or Y.

TABLE 34 2191 path 1 CDR-H1 CDR-H2 CDR-H3 VH (SEQ ID NO:) (SEQ ID NO:)(SEQ ID NO:) Kabat Kabat Kabat H10 M41 parent SYTMS (42)YISSGGGNTYYPDSVKG (46) HRGNPFDY (58) Extended CDR-H1 Kabat Kabat H31HFR (M91) GFTFSSYTMS YISSGGGNTYYPDSVKG (46) HRGNPFDY (43) (58) H227M427, GFTFSSYGMS YIDEGGGQTIYPDSVKG (47) HRGNPFDY M429 = M696; (44) (58)M488 = M694; M489 H228 M492 = M695; GFTFSSYSMS YIDAGGGFTIYPDSVKG (48)HRGNPFDY M493 (45) (58) H231 M501 GFTFSSYSMS HIDAGGGRTWYPDSVKG (49)HRGNPFDY (45) (58) H222 M526 GFTFSSYGMS YIDRGGGVTIYPDSVKG (50) HRGNPFDY(44) (58) H227 Kabat SYGMS (161) defined CDR- H1 X₁IX₂X₃GGGX₄TX₅YPDSVKG(156) For SEQ ID NO: 156, X₁ is H or Y; X₂ is D or S; X₃ is A, E, R, orS; and X₄ is I, W, or Y.

TABLE 35 2191 path 2 CDR-H1 CDR-H2 CDR-H3 VH (SEQ ID NO:) (SEQ ID NO:)(SEQ ID NO:) Kabat Kabat Kabat H10 M41 parent SYTMS (42)YISSGGGNTYYPDSVKG HRGNPFDY (58) (46) Extended CDR- H1 Kabat Kabat H39HFR; M131; GFTFSSYTMS YISSGGGNTYYPDSVKG HRGNPFDY (58) M136-138 (43) (46)H40 M132; M139; GFTFSSYTMS AIDHGGGRTYYPDSVKG HRGNPFDY (58) M140 (43)(51) H41 GFTFSSYTMS AIDHGGGRTWYPDSVKG HRGNPFDY (58) (43) (52) H79 M133;GFTFSSYTMS AIDHGGGQTLYPDSVKG HRGNPFDY (58) M141 = M707; (43) (53) M142H145 GFTFSSYTMS YISGGGGQTLYPDSVKG HRGNPFDY (58) (43) (54) H190GFTFSSYTMS TIDRGGGSTWYPDSVKG HRGNPFDY (58) (43) (55) H191 M134; M143;GFTFSSYTMS AIDGGGGATYYPDSVKG HRGNPFDY (58) M144; (43) (56) H192M135; M145; GFTFSSYTMS VIDHGGGSTHYPDSVKG HRGNPFDY (58) M146; (43) (57)M408 = M708 X₁IX₂X₃GGGX₄TX₅YPDSVKG (157) For SEQ ID NO: 157, X₁ is A, T,V, or Y; X₂ is D or S; X₃ is G, H, R, or S; X₄ is A, N, Q, R, or S; andX₅ is H, L, W, or Y.

TABLE 36 2191 path 1 CDR-L1 CDR-L2 CDR-L3 (SEQ ID NO:) (SEQ ID NO:)(SEQ ID NO:) VL Kabat Kabat Kabat L20 M41 parent RASKSVSTSGYSFMHLASNLES (67) QHSRELPWT (75) mouse (59) L42 HFR (M91) RASKSVSTSGYSFMHLASNLES (67) QHSRELPWT (75) (59) L244 M427 RASKSVSAWGYSFMH VASRLES (68)QHSRELPWT (75) (60) L245 M429 = M696 RASKSVSHVRWSFMH LASKLES (69)QHSRELPWT (75) (61) L249 M488 = M694; RASKSVSEGRWSFMH VASRLES (68)QHSRELPWT (75) M492 = M695; (62) M526 L250 M489; M493; RASKSVSLDRWSFMHLASNLES (67) QHSRELPWT (75) M501 (63) RASKSVSX₁X₂X₃X₄SFMH (158) For SEQID NO: 158, X₁ is A, E, H, L, T, or Y; X₂ is D, G, I, S, V, or W; X₃ isG or R; and X₄ is W or Y.

TABLE 37 2191 path 2 CDR-L2 (SEQ CDR-L3 (SEQ ID ID NO:) NO:) VLCDR-L1 (SEQ ID NO:) Kabat Kabat Kabat L20 M41 parentRASKSVSTSGYSFMH (59) LASNLES (67) QHSRELPWT (75) mouse L59RASKSVSTSGYSFMH (59) VGNRLED (70) QHSRELPWT (75) L62RASKSVSTSGYSFMH (59) VGDRRQE (71) QHSRELPWT (75) L124RASKSVSTSGYSFMH (59) VGSRMAF (72) QHSRELPWT (75) L131RASKSVSTSGYSFMH (59) VGDRANW (73) QHSRELPWT (75) L137 M138;RASKSVSTSGYSFMH (59) VGSRLDY (74) QHSRELPWT (75) M408 = M708 L160M137; M160; RASKSVSYVRWSFMH (64) LASNLES (67) QHSRELPWT (75) M142; M143;M146 L172 RASKSVSHIRWSFMH (65) LASNLES QHSRELPWT (75) L173 M136; M139;RASKSVSHVRWSFMH (66) LASNLES QHSRELPWT (75) M141 = M707; M144; M145Protein and Antibody Variable Region Sequences

TABLE 38 SEQ ID Features NO Clone Sequence (CDR sequences underlined)or Origin Comments 174 Human TPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRECD: 1-173, CD27 ECD KAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVR His6NCTITANAECACRNGWQCRDKECTECDPLPNPSLTARSSQALSPHPQPTHLPYVSEMLEARTAGHMQTLADFRQ LPARTLSTHWPPQRSLCSSDFIRILHHHHHH175 Human TPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHR ECD: 1-121, CD27 ECDKAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVR cleavage truncatedNCTITANAECACRNGWQCRDKECTECDPLPNPSLTAR site, His6SSQALSPHPQLEVLFQGPHHHHHH 176 Human TPAPKSCPERHYWAQGKLCCQMCEPGTFLVKDCDQHRECD: 1-101 Proteos CD27 ECD KAAQCDPCIPGVSFSPDHHTRPHCESCRHCNSGLLVRtruncated NCTITANAECACRNGWQCRDKECTECDGGHHHH 150 HumanMPEEGSGCSVRRRPYGCVLRALVPLVAGLVICLVVCI ECD CD70 ECDQRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCTNLTGTLLPSRNTDETFF GVQWVRP 76 C2186QVQLQQPGAELVKPGASVKLSCKASGYTFTNYWMNWV Murine HeavyKQRPGRGLEWIGRIHPSDSETHYNQNFKSKATLTVDK ChainSSSTAYIQLSSLTSEDSAVYYCARPVLYGDYGFPCWG (HC) QGTLVTVSA Variable Region 77C2186 DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYM Murine LightNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTD ChainFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK (LC) Variable Region 78 C2192QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWV Murine HeavyKQRPGKGLEWIGRIYPGDGDTNYNGKFKGKATLTADK ChainSSSTAYMQLSSLTSEDSAVYFCARRWDGGNYFFDYWG (HC) QGTTLTVSS Variable Region 79C2192 DIVMTQSHKFMSTSVGDRVSITCMASQDVGTAVAWYQ Murine LightRRPGQSPKLLIYWTSTRHTGVPDRFTGSGSGTDFTLT ChainISNVQSEDLADYFCQQYSSYPLTFGSGTKLEIK (LC) Variable Region 80 C2177QVQLQQSGPELVKPGASVKISCKASGYAFSSSWMNWV H7 Murine HeavyKQRPGKGLEWIGRIYPGDGDTNYNGKFKGKATLTADK ChainSSSTAYMQLSSLTSEDSAVYFCARSDYYGDYGFAYWG (HC) QGTLVTVSA Variable Region 81C2177 DIVLTQSPASLAVSLGQRATISCKASQSVDYAGDSYM L18 Murine LightNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTD ChainFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK (LC) Variable Region 82 L35DIVMTQSPDSLAVSLGERATINCKASQSVDYAGDSYM 4-1/2 HFRNWYQQKPGQPPKLLIYAASNLESGVPDRFSGSGSGTD selectedFTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIK for C2177 AM path 1 83 L255DIVMTQSPDSLAVSLGERATINCKASQSVDYAGDSFM VL inNWYQQKPGQPPKLLIYAASNLESGVPDRFSGSGSGTD M584,FTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIK M600 AM clones; in M678, M680 PTMvariants 84 L256 DIVMTQSPDSLAVSLGERATINCKASQSVDYAGDSWM C2177NWYQQKPGQPPKLLIYVASNLESGVPDRFSGSGSGTD affinityFTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIK maturation path 1 85 L257DIVMTQSPDSLAVSLGERATINCKASQSVDYAGDSWM VL inNWYQQKPGQPPKLLIYTASNLESGVPDRFSGSGSGTD M558,FTLTISSLLAEDVAVYYCQQSNEDPYTFGQGTKLEIK M596 AM clones 86 L258DIVMTQSPDSLAVSLGERATINCKASQSVDYAGSSFM C2177NWYQQKPGQPPKLLIYTASNLESGVPDRFSGSGSGTD affinityFTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIK maturation path 1 87 L260DIVMTQSPDSLAVSLGERATINCKASQSVDWAGHSWM C2177NWYQQKPGQPPKLLIYTASNLESGVPDRFSGSGSGTD affinityFTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIK maturation path 1 88 L261DIVMTQSPDSLAVSLGERATINCKASQSVDYAGSSFM C2177NWYQQKPGQPPKLLIYEASNLESGVPDRFSGSGSGTD affinityFTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIK maturation path 1 89 L267DIVMTQSPDSLAVSLGERATINCKASQSVDYAGESFM VL inNWYQQKPGQPPKLLIYAASNLESGVPDRFSGSGSGTD M703,FTLTISSLQAEDVAVYYCQQSNEDPYTFGQGTKLEIK M706 PTM variants 90 L36DIQMTQSPSSLSASVGDRVTITCKASQSVDYAGDSYM O12/2 HFRNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTD selectedFTLTISSLQPEDFATYYCQQSNEDPYTFGQGTKLEIK for C2177 AM path 2 91 L216DIQMTQSPSSLSASVGDRVTITCKASQSVDYFSESYM C2177 AMNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTD path 2FTLTISSLQPEDFATYYCQQSNEDPYTFGQGTKVEIK 92 L217DIQMTQSPSSLSASVGDRVTITCKASQSVDYAHESFM VL inNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTD M166 AMFTLTISSLQPEDFATYYCQQSNEDPYTFGQGTKVEIK clone 93 L218DIQMTQSPSSLSASVGDRVTITCKASQSVDYFGDSLM C2177 AMNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTD path 2FTLTISSLQPEDFATYYCQQSNEDPYTFGQGTKVEIK 94 L219DIQMTQSPSSLSASVGDRVTITCKASQSVDYAGDSFM VL inNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTD M160,FTLTISSLQPEDFATYYCQQSNEDPYTFGQGTKVEIK M169 AM clones 95 L219 PTMDIQMTQSPSSLSASVGDRVTITCKASQSVDYAGESFM VL inNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTD M668,FTLTISSLQPEDFATYYCQQSNEDPYTFGQGTKVEIK M669, M670, M671 PTM variants 96L266 DIQMTQSPSSLSASVGDRVTITCKASQSVDYAGeSFM VL inNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTD M668,FTLTISSLQPEDFATYYCQQSNEDPYTFGQGTKVEIK M669, M670, M671 PTM variants 97L220 DIQMTQSPSSLSASVGDRVTITCKASQSVDYAGDSYM VL inNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTD M158 AMFTLTISSLQPEDFATYYCQQSNEDPYTFGQGTKVEIK clone 98 L221DIQMTQSPSSLSASVGDRVTITCKASQSVDYFRTSFM C2177 AMNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTD path 2FTLTISSLQPEDFATYYCQQSNEDPYTFGQGTKVEIK 99 L222DIQMTQSPSSLSASVGDRVTITCKASQSVDYVGTSFM C2177 AMNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTD path 2FTLTISSLQPEDFATYYCQQSNEDPYTFGQGTKVEIK 100 L223DIQMTQSPSSLSASVGDRVTITCKASQSVDYWSDSFM C2177 AMNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTD path 2FTLTISSLQPEDFATYYCQQSNEDPYTFGQGTKVEIK 101 L224DIQMTQSPSSLSASVGDRVAITCKASQSVDYYNSSFM C2177 AMNWYQQKPGKAPKLLIYAASNLESGVPSRFSGSGSGTD path 2FTLTISSLQPEDFATYYCQQSNEDPYTFGQGTKVEIK 102 H24EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV IGHV5-51/4 HFRRQMPGKGLEWMGRIYPGDGDTNYNGKFKGQVTISADK selectedSISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG C2177 AM QGTLVTVSS path 2 103 H221EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV VH inRQMPGKGLEWMGRIYSGDGDTNYNGKFKGQVTISADK M169 AMSISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG clones QGTLVTVSS 104 H257EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV VH inRQMPGKGLEWMGRIYSGDaDTNYaqKFKGQVTISADK M670 PTMSISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG variant QGTLVTVSS 105 H258EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV VH inRQMPGKGLEWMGRIYSGDaDTNYNqKFKGQVTISADK M671 PTMSISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG variant QGTLVTVSS 106 H25QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWV IGHV1-46/4 HFRRQAPGQGLEWMGRIYPGDGDTNYNGKFKGRVTMTRDT selectedSTSTVYMELSSLRSEDTAVYYCARSDYYGDYGFAYWG for QGTLVTVSS C2177 AM, path 2 107H196 QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWV VH inRQAPGQGLEWMGRIYSGDGDTNYNGKFKGRVTMTRDT M158,STSTVYMELSSLRSEDTAVYYCARSDYYGDYGFAYWG M160 AM QGTLVTVSS clones 108 H255QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWV VH inRQAPGQGLEWMGRIYSGDADTNYAQKFKGRVTMTRDT M668,STSTVYMELSSLRSEDTAVYYCARSDYYGDYGFAYWG M672 PTM QGTLVTVSS variants 109H256 QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWV VH inRQAPGQGLEWMGRIYSGDADTNYNQKFKGRVTMTRDT M669,STSTVYMELSSLRSEDTAVYYCARSDYYGDYGFAYWG M673 QGTLVTVSS PTM variants 110H197 QVQLVQSGAEVKKPGASVKVSCKASGYAFSSSWMNWV C2177 AMRQAPGQGLEWMGRIYQGDGDTNYNGKFKGRVTMTRDT cloneSTSTVYMELSSLRSEDTAVYYCARSDYYGDYGFAYWG QGTLVTVSS 111 H28EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV IGHV5- HFRRQMPGKGLEWMGRIYPGDGDTNYSPSFQGQVTISADK 51c/4 selectedSISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG for QGTLVTVSS C2177 affinitymaturation (AM) path 1 112 H236 EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWVVH in RQMPGKGLEWMGRIYPGDGDTNYSPSFQGQVTISADK M584 AMSISTAYLQWSSLKASDTAMYYCARADYYGDYGFGYWG clone QGTLVTVSS path 1 113 H237EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV C2177 AMRQMPGKGLEWMGRIFVRDGDTNYSPSFQGQVTISADK cloneSISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG path 1 QGTLVTVSS 114 H238EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV C2177 AMRQMPGKGLEWMGRIYVGDGDTNYSPSFQGQVTISADK cloneSISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG path 1 QGTLVTVSS 115 H239EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV VH inRQMPGKGLEWMGRIYAGDGDTNYSPSFQGQVTISADK M596 AMSISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG clone QGTLVTVSS path 1 116 H240EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV VH inRQMPGKGLEWMGRIYARDGDTNYSPSFQGQVTISADK M558,SISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG M600 AM QGTLVTVSS clones, path 1117 H241 EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV C2177 AMRQMPGKGLEWMGRIYGRDGDTNYSPSFQGQVTISADK cloneSISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG path 1 QGTLVTVSS 118 H242EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV C2177 AMRQMPGKGLEWMGRIYANDGDTNYSPSFQGQVTISADK cloneSISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG path 1 QGTLVTVSS 119 H243EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV C2177 AMRQMPGKGLEWMGRIYGGDGDTNYSPSFQGQVTISADK cloneSISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG path 1 QGTLVTVSS 120 H244EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV V2177 AMRQMPGKGLEWMGRIYSGDGDTNYSPSFQGQVTISADK cloneSISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG path 1 QGTLVTVSS 121 H245EVQLVQSVAEVKKPGESLKISCKGSGYAFSSSWMNWV C2177 AMRQMPGKGLEWMGRIYSRDGDTNYSPSFQGQVTISADK cloneSISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG path 1 QGTLVTVSS 122 H259EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV VH inRQMPGKGLEWMGRIYAGDGDTAYSPSFQGQVTISADK M678SISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG PTM QGTLVTVSS mutant 123 H260EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV VH inRQMPGKGLEWMGRIYAGDGDTNYAPSFQGQVTISADK M680SISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG PTM QGTLVTVSS mutant 124 H270EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV VH inRQMPGKGLEWMGRIYAGDADTAYSPSFQGQVTISADK M703SISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG PTM QGTLVTVSS mutant 125 H272EVQLVQSGAEVKKPGESLKISCKGSGYAFSSSWMNWV VH inRQMPGKGLEWMGRIYAGDADTNYAPSFQGQVTISADK M706SISTAYLQWSSLKASDTAMYYCARSDYYGDYGFAYWG PTM QGTLVTVSS mutant

TABLE 39 SEQ Features ID Clone Sequence or Origin Comments 126 C2191EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYTMSWVRQ H10 Murine HeavyTPEKRLEWVAYISSGGGNTYYPDSVKGRFTISRDNARNT ChainLYLQMSSLRSEDTAMYYCSRHRGNPFDYWGQGTTLTVSS (HC) Variable Region 127 C2191DIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSFMHW L20 Murine LightYQQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLN ChainIHPVEEEDAATYYCQHSRELPWTFGGGTKLEIK (LC) Variable Region 128 H30EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQ IGHV3- HFRAPGKGLEWVSYISSGGGNTYYPDSVKGRFTISRDNSKNT 23/4 selectedLYLQMNSLRAEDTAVYYCAKHRGNPFDYWGQGTLVTVSS for C2191 AM path 2 129 H79EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQ VH inAPGKGLEWVSAIDHGGGQTLYPDSVKGRFTISRDNSKNT M141LYLQMNSLRAEDTAVYYCARHRGNPFDYWGQGTLVTVSS AM variant, path 2. Table 29 130H192 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQ VH inAPGKGLEWVSVIDHGGGSTHYPDSVKGRFTISRDNSKNT M408LYLQMNSLRAEDTAVYYCARHRGNPFDYWGQGTLVTVSS AM variant, path 2. Table 29 131H31 QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYTMSWIRQ IGHV3- HFRAPGKGLEWVSYISSGGGNTYYPDSVKGRFTISRDNAKNS 11/4 selectedLYLQMNSLRAEDTAVYYCARHRGNPFDYWGQGTLVTVSS for C2191 AM path 1 132 H222QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWIRQ VH inAPGKGLEWVSYIDRGGGVTIYPDSVKGRFTISRDNAKNS M526 AMLYLQMNSLRAEDTAVYYCARHRGNPFDYWGQGTLVTVSS variant, path 1 133 H227QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWIRQ VH inAPGKGLEWVSYIDEGGGQTIYPDSVKGRFTISRDNAKNS M427,LYLQMNSLRAEDTAVYYCARHRGNPFDYWGQGTLVTVSS M429, M488, M489 AM variants,path 1 134 H228 QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMSWIRQ VH inAPGKGLEWVSYIDAGGGFTIYPDSVKGRFTISRDNAKNS M492,LYLQMNSLRAEDTAVYYCARHRGNPFDYWGQGTLVTVSS M493 AM variants, path 1 135H231 QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMSWIRQ VH inAPGKGLEWVSHIDAGGGRTWYPDSVKGRFTISRDNAKNS M501 AMLYLQMNSLRAEDTAVYYCARHRGNPFDYWGQGTLVTVSS variant, path 1 136 H232QVQLVESGGGLVKPGGSLRLSCAASGFTFSSYPMSWIRQ C2191APGKGLEWVSHIATGGGNTYYPDSVKGRFTISRDNAKNS AMLYLQMNSLRAEDTAVYYCARHRGNPFDYWGQGTLVTVSS clone, path 1 137 L40DIQMTQSPSSLSASVGDRVTITCRASKSVSTSGYSFMHW IGKVO12/1 HFRYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLT selectedISSLQPEDFATYYCQHSRELPWTFGQGTKVEIK for C2191 AM, path 2 138 L137DIQMTQSPSSLSASVGDRVTITCRASKSVSTSGYSFMHW VL inYQQKPGKAPKLLIYVGSRLDYGVPSRFSGSGSGTDFTLT M408ISSLQPEDFATYYCQHSRELPWTFGQGTKVEIK AM variant, path 2. Table 29 139 L173DIQMTQSPSSLSASVGDRVTITCRASKSVSHVRWSFMHW VL inYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLT M141ISSLQPEDFATYYCQHSRELPWTFGQGTKVEIK AM variant, path 2. Table 29 140 L42DIQMTQSPSSLSASVGDRVTITCRASKSVSTSGYSFMHW IGKVO8/1 HFRYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTFT selectedISSLQPEDIATYYCQHSRELPWTFGQGTKVEIK for C2191 AM path 1 141 L244DIQMTQSPSSLSASVGDRVTITCRASKSVSAWGYSFMHW VL inYQQKPGKAPKLLIYVASRLESGVPSRFSGSGSGTDFTFT M427 AMISSLQPEDIATYYCQHSRELPWTFGQGTKVEIK variant, path 1 142 L245DIQMTQSPSSLSASVGDRVTITCRASKSVSHVRWSFMHW VL inYQQKPGKAPKLLIYLASKLESGVPSRFSGSGSGTDFTFT M429 AMISSLQPEDIATYYCQHSRELPWTFGQGTKVEIK variant, path 1 143 L249DIQMTQSPSSLSASVGDRVTITCRASKSVSEGRWSFMHW VL inYQQKPGKAPKLLIYVASRLESGVPSRFSGSGSGTDFTFT M488,ISSLQPEDIATYYCQHSRELPWTFGQGTKVEIK M492, M526 AM variants, path 1 144L250 DIQMTQSPSSLSASVGDRVTITCRASKSVSLDRWSFMHW VL inYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTFT M489,ISSLQPEDIATYYCQHSRELPWTFGQGTKVEIK M493, M501 AM variants, path 1 145 H39EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQ HFR; VHAPGKGLEWVSYISSGGGNTYYPDSVKGRFTISRDNSKNT inLYLQMNSLRAEDTAVYYCARHRGNPFDYWGQGTLVTVSS M131; M136-138 AM clones, path 2146 H40 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQ VH inAPGKGLEWVSAIDHGGGRTYYPDSVKGRFTISRDNSKNT M132;LYLQMNSLRAEDTAVYYCARHRGNPFDYWGQGTLVTVSS M139; M140 AM clones, path 2 147H191 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYTMSWVRQ VH inAPGKGLEWVSAIDGGGGATYYPDSVKGRFTISRDNSKNT M134;LYLQMNSLRAEDTAVYYCARHRGNPFDYWGQGTLVTVSS M143; M144; AM clones, path 2148 L160 DIQMTQSPSSLSASVGDRVTITCRASKSVSYVRWSFMHW VL inYQQKPGKAPKLLIYLASNLESGVPSRFSGSGSGTDFTLT M137;ISSLQPEDFATYYCQHSRELPWTFGQGTKVEIK M160; M142; M143; M146 clones, path 2

TABLE 40 CD27 and CD70 Proteins SEQ ID NO: DESCRIPTIONFeatures, Abbreviations 149 Human CD27 1-20 Signal, 21-191propolypeptide Extracellular domain, 192-212 Transmembrane,213-260 Intracellular TPAPKSCPER HYWAQGKLCC QMCEPGTFLV KDCDQHRKAAQCDPCIPGVS FSPDHHTRPH CESCRHCNSG LLVRNCTITA NAECACRNGW QCRDKECTECDPLPNPSLTA RSSQALSPHP QPTHLPYVSE MLEARTAGHM QTLADFRQLPARTLSTHWPP QRSLCSSDFI RILVIFSGMF LVFTLAGALF LHQRRKYRSNKGESPVEPAE PCRYSCPREE EGSTIPIQED YRKPEPACSP 150 Human CD701-17 intracellular, propolypeptide 18-38 transmembrane,and 39-193 extracellular MPEEGSGCSV RRRPYGCVLR AALVPLVAGL VICLVVCIQRFAQAQQQLPL ESLGWDVAEL QLNHTGPQQD PRLYWQGGPA LGRSFLHGPE LDKGQLRIHRDGIYMVHIQV TLAICSSTTA SRHHPTTLAV GICSPASRSI SLLRLSFHQGCTIASQRLTP LARGDTLCTN LTGTLLPSRN TDETFFGVQW VRP

TABLE 41 Antibody constant region sequences SEQ ID Description SequenceNO: Heavy chain astkgpsvfplapcsrstsestaalgclvkdyfpepvtvswn 159human IgG4 sgaltsgvhtfpavlqssglyslssvvtvpssslgtktytcn Ala/Ala Servdhkpsntkvdkrveskygppcppcpapeaaggpsvflfppk to Propkdtlmisrtpevtcvvvdvsqedpevqfnwyvdgvevhnak constanttkpreeqfnstyrvvsvltvlhqdwingkeykckvsnkglps regionsiektiskakgqprepqvytlppsqeemtknqvsltclvkgfypsdiavewesngqpennykttppvldsdgsfflysrltvdksrwqegnvfscsvmhealhnhytqkslslslgk Light chainrtvaapsvfifppsdeqlksgtasvvcllnnfypreakvqwk 160 human kappavdnalqsgnsqesvteqdskdstyslsstltlskadyekhkv constantyacevthqglsspvtksfnrgec region

TABLE 42 Antibody nucleotide sequences SEQ ID Description Sequence NO:Light Chain GACATCCAGATGACCCAGAGCCCCAGCAGCCTGAGCGCCAGCGTGGGCGACCGGG 163encoding TGACCATCACCTGCCGGGCCAGCAAGAGCGTGAGCGAGGGGCGATGGAGCTTCATsequence GCACTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACGTGGCC(encodes the AGCAGACTGGAGAGCGGCGTGCCCAGCCGGTTCAGCGGCAGCGGCAGCGGCACCGlight chain ACTTCACCTTCACCATCAGCAGCCTGCAGCCCGAGGACATCGCCACCTACTACTGsequence of CCAGCACAGCCGGGAGCTGCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCSEQ ID NO: AAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGT 160)TGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT HeavyCAGGTGCAGCTGGTGGAGAGCGGCGGCGGCCTGGTGAAGCCCGGCGGCAGCCTGC 164 ChainGGCTGAGCTGCGCCGCCAGCGGCTTCACCTTCAGCAGCTACGGGATGAGCTGGAT encodingCCGGCAGGCCCCCGGCAAGGGCCTGGAGTGGGTGAGCTACATCGATGAGGGCGGC sequenceGGCCAGACCATCTACCCCGACAGCGTGAAGGGCCGGTTCACCATCAGCCGGGACA (encodes theACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGC heavy chainCGTGTACTACTGCGCCCGGCACCGGGGCAACCCCTTCGACTACTGGGGCCAGGGC sequence ofACCCTGGTGACCGTGAGCAGCGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGG SEQ ID NO:CGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAA 159)GGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAAACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGGCCGCCGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTG TCTCTGGGTAAA

What is claimed:
 1. A method of in vitro inhibiting T-cell mediatedB-cell antibody production, comprising contacting a portion of humanT-cells with an effective amount of a human CD27 antibody orantigen-binding fragment thereof comprising a light chain variableregion and a heavy chain variable region, said light chain variableregion comprising: a complementarity determining region light chain 1(CDRL1) amino acid sequence selected from the group consisting of SEQ IDNOs: 59-66 and 158; a CDRL2 amino acid sequence selected from the groupconsisting of SEQ ID NOs: 67-74; and a CDRL3 amino acid sequence of SEQID NO: 75; said heavy chain variable region comprising: a CDRH1 aminoacid sequence selected from the group consisting of SEQ ID NOs: 42-45and 161; a CDRH2 amino acid sequence selected from the group consistingof SEQ ID NOs: 46-57, 156, and 157; and a CDRH3 amino acid sequence ofSEQ ID NO: 58, and the antibody or antigen binding portion thereofinhibits the activation of CD27 on said human T-cells in the presence ofhuman CD70 protein.
 2. The method of claim 1, wherein the contactingstep comprises contacting the T-cells with an effective amount of ahuman CD27 antibody or antigen-binding fragment thereof comprising alight chain variable region and a heavy chain variable region, saidlight chain variable region comprising: a CDRL1 amino acid sequence ofSEQ ID NO: 62; a CDRL2 amino acid sequence of SEQ ID NO: 68; and a CDRL3amino acid sequence of SEQ ID NO: 75, and said heavy chain variableregion comprising: a CDRH1 amino acid sequence selected from the groupconsisting of SEQ ID NOs: 44 and 161; a CDRH2 amino acid sequence of SEQID NO: 47; and a CDRH3 amino acid sequence of SEQ ID NO:
 58. 3. Themethod of claim 1, wherein the contacting step comprises contacting theT-cells with an effective amount of a human CD27 antibody orantigen-binding fragment thereof comprising a light chain variable aminoacid sequence of SEQ ID NO: 143 and a heavy chain variable amino acidsequence of SEQ ID NO:
 133. 4. The method of claim 3, wherein theantibody further comprises an IgG4 heavy chain constant region and anIgG4 light chain constant region.
 5. The method of claim 4, wherein theheavy chain constant region comprises the amino acid sequence of SEQ IDNO:159 and the light chain constant region comprises the amino acidsequence of SEQ ID NO:160.